Methods of characterizing a urine sample

ABSTRACT

This disclosure relates to methods of characterizing a urine sample from a subject.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/406,692, filed on Oct. 11, 2016, the contents of which areincorporated by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to methods of urine testing.

BACKGROUND

Urine drug testing is a commonly used tool to detect a subject's use ofdrugs, both legal (e.g., controlled substances) and illegal. During thelast half century the use of urine drug testing has been used throughoutthe military, in the public and private workplace, in courts, and inmedical clinics and care centers. The urine drug tests are usedprimarily to detect illegal or banned substances in a subject's system.In the clinical setting, physicians test their patients to determine iftheir patients are adhering to their prescriptions. Urine drug testinghas become a routinely used effective tool in the assessment and ongoingmanagement of patients who are treated with controlled substances for,e.g., chronic pain. The urine drug testing results provide confirmationof the agreed-upon treatment plan and diagnose relapse or drug abuse.

The results of a urine drug test can have serious consequences for apatient including termination of prescription. In fear of the possibleconsequences, patients have developed a variety of methods to cheat bysubstituting their own urine sample with synthetic orchemically-adulterated urines. Patients who “cheat” a urine drug test byusing chemically-adulterated samples or synthetic urine present aproblem for the health care because the ongoing care plan will not bebased on accurate information. Currently, the best method for validatingthat a patient's sample is in fact their own is by observation duringsample collection—which is not always possible.

SUMMARY

The present invention focuses on methods developed to characterize aurine sample from a subject (e.g., used in association with drug testingor to achieve quality control). In view of this discovery, providedherein are methods of characterizing a urine sample that include: (a)providing a urine sample from a subject, determining one or more of thelight absorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of the urine sample at two or more wavelengths oflight; (c) applying one or more eigenvectors derived from a principlecomponent algorithm to a standardized dataset derived from thedetermined light absorption, luminescence, phophophorescence,fluorescence, and chemiluminescence of the urine sample to generate aUrine Characterization Index (UCI) including one or more values forcorresponding principle component(s); and (d) charactering a urinesample as a natural urine sample, a lyophilized urine sample, a dilutednatural urine sample, a synthetic urine sample, or achemically-adulterated urine sample based on the UCI. Also provided aremethods of characterizing a urine sample from a subject that include:(a) providing a urine sample collected from a subject; determining theabsence at a first wavelength (A1) and the absorbance at a secondwavelength (A2) of the urine sample, where the first wavelength is fromabout 230 nm to about 250 nm, and the second wavelength is from about260 nm to 340 nm; (c) applying an algorithm to the determined A1 and thedetermined A2 to generate a Urine Characterization Score (UCS), wherethe algorithm includes a ratio of the determined A1 to the determinedA2; and (d) characterizing a urine sample as a natural urine sample, achemically-adulterated urine sample, a lyophilized urine sample, adiluted natural urine sample, or a synthetic urine sample based on theUCS.

Provided herein are methods of characterizing a urine sample from asubject that include: (a) providing a urine sample from a subject; (b)determining one or more (e.g., two, three, four, or five) of the lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of the urine sample at two or more wavelengths oflight; (c) applying one or more eigenvectors derived from a principlecomponent algorithm to a standardized dataset derived from thedetermined light absorption, luminescence, phosphorescence,fluorescence, and chemiluminescence of the urine sample to generate aUrine Characterization Index (UCI) including one or more values forcorresponding principle component(s); and (d) characterizing a urinesample as a natural urine sample, a lyophilized urine sample, a dilutednatural urine sample, a synthetic urine sample, or achemically-adulterated urine sample based on the UCI. In someembodiments of these methods, the chemically-adulterated urine is asurfactant-adulterated urine. In some embodiments of these methods, step(b) includes determining the light absorption of the urine sample. Insome embodiments of these methods, step (b) includes determining thelight absorption of one or more of ultraviolet light wavelength(s),visible light wavelength(s), near-infrared light wavelength(s), andinfrared absorption light wavelengths. In some embodiments of thesemethods, step (b) includes determining the light absorption of the urinesample at wavelengths of about 200 nm to about 1000 nm. In someembodiments of these methods, step (b) includes determining the lightabsorption of the urine sample at wavelengths of about 200 nm to about340 nm.

In some embodiments of these methods, step (c) includes applying aneigenvector derived from the principle component algorithm to a datasetderived from the determined light absorption to generate a value for oneprinciple component. In some embodiments of these methods, step (c)includes applying two eigenvectors derived from the principle componentalgorithm to a dataset derived from the determined light absorption,luminescence, phosphorescence, fluorescence, chemiluminescence, orbioluminescence to generate values in two-dimensional principlecomponent space. In some embodiments of these methods, step (c) includesapplying two eigenvectors derived from the principle component algorithmto a dataset derived from the determined light absorption to generatevalues in two-dimensional principle component space. In some embodimentsof these methods, step (c) includes applying three eigenvectors derivedfrom the principle component algorithm to a dataset derived from thedetermined light absorption, luminescence, phosphorescence,fluorescence, chemiluminescence, or bioluminescence to generate valuesin three-dimensional principle component space. In some embodiments ofthese methods, step (c) includes applying three eigenvectors derivedfrom the principle component algorithm to a dataset derived from thedetermined light absorption to generate values in three-dimensionalprinciple component space. In some embodiments of these methods, step(c) includes applying four eigenvectors derived from the principlecomponent algorithm to a dataset derived from the determined lightabsorption, luminescence, phosphorescence, fluorescence,chemiluminescence, or bioluminescence to generate values infour-dimensional principle component space. In some embodiments of thesemethods, step (c) includes applying four eigenvectors derived from theprinciple component algorithm to a dataset derived from the determinedlight absorption to generate values in four-dimensional principlecomponent space.

In some embodiments of these methods, step (d) includes characterizing aurine sample through the use of a clustering algorithm. In someembodiments of these methods, the clustering algorithm is a hierarchicalclustering algorithm, a k-means clustering algorithm, or a statisticaldistribution model. In some embodiments of these methods, step (d)includes characterizing a urine sample by performing regression analysison the values of principle components. In some embodiments of thesemethods, step (d) includes comparing the UCI of the sample to a UCI of anatural urine sample, a chemically-adulterated urine sample, alyophilized urine sample, a diluted natural urine sample, or a syntheticurine sample.

Also provided herein are methods of characterizing a urine sample from asubject that include: (a) providing a urine sample collected from asubject; (b) determining the absorbance at a first wavelength (A1) andthe absorbance at a second wavelength (A2) of the urine sample, wherethe first wavelength is from about 230 nm to 250 nm, and the secondwavelength is from about 260 nm to 340 nm; (c) applying an algorithm tothe determined A1 and the determined A2 to generate a UrineCharacterization Score (UCS), where the algorithm includes a ratio ofthe determined A1 to the determined A2; and (d) characterizing a urinesample as a natural urine sample, a chemically-adulterated urine sample,a lyophilized urine sample, a diluted natural urine sample, or asynthetic urine sample based on the UCS. In some embodiments of thesemethods, the UCS is generated using the Formula (XI): UCS=A1/A2. In someembodiments of these methods, the UCS is generated using the Formula(XII): UCS=10×log (A1/A2). In some embodiments of these methods, thefirst wavelength is 240 nm and the second wavelength is 280 nm. In someembodiments of these methods, step (d) includes charactering the urinesample as a synthetic urine sample if UCS is greater than 0.8. In someembodiments of these methods, step (d) includes charactering the urinesample as a synthetic urine sample if the UCS is less than 0.25.

In some embodiments of any of the methods described herein, the urinesample is characterized as a natural urine sample and the method furtherincludes: (e) performing an assay to determine the level of one or moredrug metabolites in the urine sample. Some embodiments of these methodsfurther include: (f) identifying a subject having an elevated level ofone or more drug metabolites in the urine sample as compared to areference level of the one or more drug metabolites, where the drugmetabolites are metabolites of an illegal or controlled substance; and(g) admitting the subject into a drug dependency program, ceasingadministration of the controlled substance to the subject, or reducingthe dose and/or frequency of administration of the controlled substanceto the subject. In some embodiments of these methods, the drugdependency program includes administering to the subject in step (g) adrug replacement therapy.

Some embodiments of any of the methods described herein further include:(e) selecting a subject having a urine sample characterized in step (d)as a surfactant-adulterated urine sample; and (f) obtaining anadditional urine sample from the selected subject. Some embodiments ofany of the methods described herein further include: (e) selecting asubject having a urine sample characterized in step (d) as a lyophilizedurine sample; and (f) obtaining an additional urine sample from theselected subject. Some embodiments of any of the methods describedherein further include: (e) selecting a subject having a urine samplecharacterized in step (d) as a diluted natural urine sample; and (f)obtaining an additional urine sample from the selected subject. Someembodiments of any of the methods described herein further include: (e)selecting a subject having a urine sample characterized in step (d) as asynthetic urine sample; and (f) obtaining an additional urine samplefrom the selected subject.

In some embodiments of any of the methods described herein, theadditional urine sample is obtained through a witnessed urine test. Someembodiments of any of the methods described herein further include: (g)performing an assay to determine the level of one or more drugmetabolites in the additional urine sample. Some embodiments of any ofthe methods described herein further include: (h) identifying a subjecthaving an elevated level of one or more drug metabolites in theadditional urine sample as compared to a reference level of the one ormore drug metabolites, where the drug metabolites are metabolites of anillegal or controlled substance; and (i) admitting the subject into adrug dependency program, ceasing administration of the controlledsubstance to the subject, or reducing the dose and/or frequency ofadministration of the controlled substance to the subject. In someembodiments of any of the methods described herein, the drug dependencyprogram includes administering to the subject in step (i) a drugreplacement therapy.

Some embodiments of any of the methods described herein further include:(e) selecting a subject having a urine sample characterized in step (d)as a surfactant-adulterated urine sample; (f) obtaining a sampleincluding blood, serum, hair, or plasma from the subject; and (g)performing an assay to determine the level of one or more drugmetabolites in the sample from step (f). Some embodiments of any of themethods described herein further include: (e) selecting a subject havinga urine sample characterized in step (d) as a lyophilized urine sample;(f) obtaining a sample including blood, serum, hair, or plasma from thesubject; and (g) performing an assay to determine the level of one ormore drug metabolites in the sample from step (f). Some embodiments ofany of the methods described herein further include: (e) selecting asubject having a urine sample characterized in step (d) as a dilutednatural urine sample; (f) obtaining a sample including blood, serum,hair, or plasma from the subject; and (g) performing an assay todetermine the level of one or more drug metabolites in the sample fromstep (f). Some embodiments of any of the methods provided herein furtherinclude: (e) selecting a subject having a urine sample characterized instep (d) as a synthetic urine sample; (f) obtaining a sample includingblood, serum, hair, or plasma from the subject; and (g) performing anassay to determine the level of one or more drug metabolites in thesample from step (f). Some embodiments of any of the methods describedherein further include: (h) identifying a subject having an elevatedlevel of one or more drug metabolites in the sample from step (f) ascompared to a reference level of the one or more drug metabolites, wherethe drug metabolites are metabolites of an illegal or controlledsubstance; and (i) admitting the subject into a drug dependency program,ceasing administration of the controlled substance to the subject, orreducing the dose or frequency of administration of the controlledsubstance to the subject. In some embodiments of any of the methodsdescribed herein, the drug dependency program includes administering tothe subject in step (i) a drug replacement therapy.

Some embodiments of any of the methods described herein further includerecording the characterization of the urine sample in the subject'smedical record. In some embodiments of any of the methods describedherein, the subject's medical record is a computer readable medium. Someembodiments of any of the methods described herein further includenotifying the subject's insurance provider, employer, or potentialfuture employer of the characterization of the urine sample. Someembodiments of any of the methods described herein further includenotifying a pharmacist or a medical professional of the characterizationof the urine sample.

In some embodiments of any of the methods described herein, the subjecthas not been diagnosed as having an illegal or controlled substanceaddiction. In some embodiments of any of the methods described herein,the subject has been identified as having an illegal or controlledsubstance addiction. In some embodiments of any of the methods describedherein, the subject is being treated on an outpatient basis for anillegal or controlled substance addiction.

In some embodiments of any of the methods described herein, thedetermining in step (b) is performed using high throughput processing.Some embodiments of any of the methods described herein further includeamplifying and sequencing nucleic acid present in the urine sample. Someembodiments of any of the methods described herein further includedetecting one or more of statherin, alpha-amylase, lingual lipase, andlysozyme in the urine sample.

As used herein, the word “a” before a noun represents one or more of theparticular noun. For example, the phrase “a urine sample” represents“one or more urine samples.”

The term “subject” means a vertebrate, including any member of the classmammalia, including humans, sports or pet animals, such as horse (e.g.,race horse) or dog (e.g., race dogs), and higher primates. In preferredembodiments, the subject is a human.

The term “natural urine sample” is a urine produced by the body of amammal (e.g., human).

The term “lyophilized urine sample” is a natural urine sample that hasbeen lyophilized and reconstituted in a fluid (e.g., an aqueoussolution, e.g., water).

The term “diluted natural urine sample” is a natural urine sample thathas been diluted with a fluid (e.g., an aqueous solution, e.g., water)that is not a natural urine sample.

The term “synthetic urine sample” is art known and means a syntheticliquid that is not produced by the body of a mammal (e.g., human) thatis meant to substitute for urine produced by the body of a mammal (e.g.,a human). As is known in the art, synthetic urine is commerciallyavailable from a number of vendors. As is known in the art, syntheticurine typically contains nitrogenous waste products of synthetic origin(e.g., urea, creatinine, and/or uric acid), and inorganic saltsincluding, e.g., potassium chloride, calcium chloride, sodium chloride,magnesium chloride, calcium chloride, sodium sulfate, ammoniumdiphosphate, potassium diphosphate, sodium phosphate, and/or sodiumdiphosphate. Synthetic urine is also known, on occasion, to be fortifiedwith, e.g., preservative agents (e.g., sodium azide), hormones (e.g.,estrogen), and/or coloring agents (e.g., synthetic dyes, B vitamins,and/or natural pigments (e.g., (β-carotene)).

The term “chemically-adulterated urine sample” is a natural urine samplethat has been contacted with a chemical substance to alter one or morebiophysical properties of the natural urine sample. For example, achemically-adulterated urine sample can be contacted with a chemicalsubstance that alters the absorbance of the natural urine sample at oneor more wavelengths of light. For example, a chemically-adulteratedurine sample can be contacted with a surfactant to alter one or morebiophysical properties of the natural urine sample.

As used herein, the term “principal component analysis” or “principalcomponent algorithm” refers to a statistical method that uses anorthogonal transformation to convert a set of two or more observationsof two or more possibly correlated variables into a set of two or morevalues of linearly uncorrelated variables called principal components.Principle component analysis finds the two or more principal componentsof the dataset and transforms the data into a new, lower-dimensionalsubspace. The first principle component, which can be represented by aneigenvector, mathematically corresponds to a direction in the originaln-dimensional space, so that the first principal component accounts foras much of the variance in the data as possible, and each succeedingprinciple component accounts for as much of the remaining variance aspossible.

As used herein, the term “principal component space” refers to asubspace in which the original data (e.g., one or more of the lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of a urine sample at two or more wavelengths of light)has been transformed into.

As used herein, the term “eigenvector” refers to a vector which, whenacted on by a particular linear transformation, produces a scalarmultiple of the original vector. The scalar in question refers to aneigenvalue corresponding to a particular eigenvector.

The term “drug metabolite” is art known and means a break-down productof a controlled or illegal substance produced by a mammal's bodyfollowing administration of the controlled or illegal substance to themammal (e.g., human). A wide variety of drugs, drug metabolites, andassays for detecting the levels of drugs and drug metabolites are knownin the art. Non-limiting examples of drugs, drug metabolites, andvendors that sell kits for determining the level of one or more drugsand drug metabolites are described herein.

The term “potential future employer” means a person or business entitythat is considering a subject for employment and that requires or asksemployment candidates to provide a urine sample for testing as part ofthe job application process. For example, a potential future employercan be a state or federal government, a medical care facility (e.g., aclinic or a hospital), a transportation company, or an airline company.

The term “controlled substance” means an agent or material that isregulated by a government (e.g., state, federal government, or agovernmental drug regulatory agency, such as the U.S. Food and DrugAdministration), but its administration to at least some persons is notillegal. For example, the dosage and frequency of administration of acontrolled substance can be regulated by a government. In some examples,certain persons in a population are warned not to consume a controlledsubstance. Non-limiting examples of controlled substances areprescription drugs and marijuana.

The term “drug replacement therapy” means administration of an agentthat mimics the pharmacological effect of a controlled or illegalsubstance but is longer acting, less potent, less toxic, and/or has animproved safety profile than the controlled or illegal substance.

The term “DNA-adulterated sample” means a urine sample (e.g., syntheticurine sample) from a subject that has been manipulated to add genomicDNA from the subject, where the added genomic DNA is from a source otherthan mammalian cells present in urine.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a graph showing absorbance spectra of natural urine samples.The absorbance characteristics can be described qualitatively as strongabsorbance throughout the ultraviolet region, particularly atwavelengths centered on 240 nm, 280 nm, and 300 nm.

FIG. 2 is a graph showing model spectra of natural urine samplesgenerated by the results from principal component analysis. Principalcomponent analysis reveals that the four primary principal componentscan be used to construct the model spectra. These model spectra are ingood agreement with the raw data shown in FIG. 1, although there is asizable reduction in the noise that does not contribute todistinguishable variance between the samples.

FIG. 3 is a Scree Plot indicating the % of total variance observed innatural urine samples, lyophilized urine samples, diluted natural urinesamples, synthetic urine samples, and chemically-adulterated urinesamples per principal component (or eigenvalue). These results indicatethat 86% of the variance in the characteristic spectra of natural urinesamples, lyophilized urine samples, diluted natural urine samples,synthetic urine samples, and chemically-adulterated urine samples can beaccounted for by the first three principle components.

FIG. 4 is a graph showing characteristic ultraviolet absorbance spectrafor lyophilized urine samples, diluted natural urine samples, syntheticurine samples, or chemically-adulterated urine samples.

FIG. 5 is a graph showing model spectra generated from the results ofprinciple component analysis. The model spectra is based upon a reduceddata set of only 4 of 15 principle components. These principlecomponents account for enough variation of absorbance spectra of thelyophilized urine samples, diluted natural urine samples, syntheticurine samples, and chemically-adulterated urine samples as shown in FIG.4 and the model spectra accurately reproduces major spectroscopicfeatures in the wavelength range of 200 to 340 nm.

FIG. 6 is a graph showing a cluster plot for natural urine samples,lyophilized urine samples, diluted natural urine samples, syntheticurine samples, and chemically-adulterated urine samples based on thethree principle components associated with the variation inspectroscopic data.

DETAILED DESCRIPTION

This disclosure relates to methods of characterizing a urine sample froma subject. Non-limiting aspects of these methods are described below. Ascan be appreciated in the art, the various aspects described below canbe used in any combination without limitation.

Subjects

In any of the methods described herein, a subject may be either a humanor a non-human animal. In some embodiments, the subject has not beendiagnosed as having an illegal or controlled substance addiction. Insome embodiments of any of the methods described herein, the subject hasbeen identified as having an illegal or controlled substance addiction(e.g., a subject that has already undergone treatment (e.g., successfulor unsuccessful treatment) for his or her illegal or controlledsubstance addiction). In some embodiments of any of the methodsdescribed herein, the subject is being treated on an outpatient basisfor an illegal or controlled substance addiction. In some embodiments,the subject is receiving inpatient treatment for his or her illegal orcontrolled substance addiction.

In some embodiments, the subject is a female (e.g., a pregnant female).In some embodiments, the subject is a male. A subject in any of themethods described herein can be a child, an adolescent, a teenager, oran adult (a subject that greater than 18 years old, e.g., greater than20 years old, greater than 25 years old, greater than 30 years old,greater than 35 years old, greater than 40 years old, greater than 45years old, greater than 50 years old, greater than 55 years old, greaterthan 60 years old, greater than 65 years old, greater than 70 years old,greater than 75 years old, greater than 80 years old, greater than 90years old, or greater than 100 years old). In any of the methodsdescribed herein, the subject may employed by the military, may be atruck driver, a train engineer, a pilot, a medical professional (e.g., aphysician, nurse, nurse's assistant, or a physician's assistant), or apharmacist. In any of the methods described herein, the subject has afamily history of illegal or controlled substance addiction. In any ofthe methods described herein, the subject can be identified aspreviously submitting a synthetic urine sample, a diluted natural urinesample, a urine sample originating from another subject, a lyophilizedurine sample, a chemically-adulterated urine sample, or an adulteratedurine sample.

Urine Samples

Some embodiments of the methods described herein can include a step ofproviding a urine sample collected from a subject. In some examples, themethods described herein can further include a step of obtaining a urinesample from a subject. A urine sample is typically obtained usingunwitnessed urine sample collection. As described herein, a urine samplecan be a natural urine sample, a synthetic urine sample, achemically-adulterated urine sample, a lyophilized urine sample, or adiluted natural urine sample, or any mixture thereof.

A urine sample can have a volume of at least 1 mL (e.g., at least 2 mL,at least 3 mL, at least 4 mL, at least 5 mL, at least 6 mL, at least 7mL, at least 8 mL, at least 9 mL, at least 10 mL, at least 12 mL, atleast 14 mL, at least 16 mL, at least 18 mL, at least 20 mL, at least 22mL, at least 24 mL, at least 26 mL, at least 28 mL, or at least 30 mL).For example, a urine sample can have a volume of between about 1 mL andabout 30 mL, between about 5 mL and about 30 mL, between about 10 mL andabout 30 mL, or between about 15 mL and about 30 mL.

In some examples of any of the methods described herein, the urinesample can be stored, e.g., for at least 1 hour (e.g., at least 6 hours,at least 12 hours, at least 1 day, at least 2 days, at least 3 days, atleast 4 days, at least 5 days, at least 6 days, or at least 7 days) at atemperature below 25° C. (e.g., at about 15° C., at about 10° C., atabout 4° C., at about 0° C., at about −20° C., at about −40° C., atabout −80° C., at about −86° C., or at about −196° C.) before any of thepresent methods are performed on the urine sample. In some embodiments,the urine sample can be centrifuged or clarified (e.g., by gravity)before any of the present methods are performed. In some embodiments,the urine sample can be filtered (e.g., to remove any mammalian cells,bacterial cells, yeast cells, or soluble or insoluble proteinaggregates) before any of the present methods are performed.

A natural urine sample may include the presence of one or more ofcellular material including intact cells, components of cellularsystems, or cellular debris originating from the mammal providing thespecimen, or originating from the growth of prokaryotic lifeformsincluding any bacterial cells or archaea, or originating from the growthof eukaryotic lifeforms including protists, fungi, or animals found tobe in urine from either infection, parasitic growth, or contaminationwith living organisms after collection. Biological properties of anatural urine sample may also include the presence of enzymatic activityarising from freely soluble enzymes in a natural urine sample, ormembrane-bound enzyme(s) adhered or imbedded to cells or cellular debrisarising from either prokaryotic or eukaryotic origin. The biologicalproperties of a natural urine sample may also include the presence orevidence of genetic material including DNA or RNA arising fromprokaryotic, eukaryotic, or viral origins or the presence or evidence ofgenetic processes including but not limited to the replication ofnucleic acids, DNA cleavage, DNA damage, DNA methylation, DNA repair,gene expression, gene regulation, mutagenesis, nucleic aciddenaturation, recombination, RNA cleavage, and viral integration.

The chemical composition of a natural urine sample may also include oneor more of the presence or concentration of 1-aliphatic acycliccompounds, aliphatic heteromonocyclic compounds, aliphaticheteropolycyclic compounds, aliphatic homomonocyclic compounds,aliphatic homopolycyclic compounds, alkaloids, and derivatives, aminoacids, peptides and analogues, aromatic heteromonocyclic compounds,aromatic heteropolycyclic compounds, aromatic homomonocyclic compounds,aromatic homopolycyclic compounds, carbohydrates and carbohydrateconjugates, homogenous metal compounds, homogenous non-metal compounds,inorganic compounds, lignans and norlignans, lipids, mixedmetal/non-metal compounds, nucleosides, nucleotides and analogs, organicacids and derivatives, organic halides, organometallic compounds,organophosphorous compounds, polyketides, tannins, bile acids,degradation or chemical-reaction, or enzymatic-reaction products of anyof the above, or other compounds both endogenous and xenobiotic in anatural urine sample occurring due to metabolic processes or otherwisefound to occur in a urine sample.

Lyophilized urine samples, diluted natural urine samples, syntheticurine samples, and chemically-adulterated urine samples do not have thesame biological properties and/or chemical composition as a naturalurine sample, and manipulation of a natural urine sample (e.g.,dilution, chemical-adulteration, and lyophilization) can change thecharacteristics of a natural urine sample. The methods described hereincan detect the differences in the biological properties and/or chemicalcomposition between a natural urine sample and a synthetic urine sample,a chemically-adulterated urine sample, a lyophilized urine sample, and adiluted natural urine sample, and can be used to accurately characterizea urine sample as a natural urine sample, a synthetic urine sample, achemically-altered urine sample, a lyophilized urine sample, or adiluted natural urine sample.

Methods of Characterizing a Urine Sample from a Subject that IncludeDetermining a Urine Characterization Score

Provided are methods of characterizing a urine sample from a subjectthat include: (a) providing a urine sample from a subject; (b)determining the absorbance at a first wavelength (A1) and the absorbanceat a second wavelength (A2) of the urine sample, where the A1 is fromabout 230 nm to about 250 nm (inclusive), and the A2 is from about 260nm to about 340 nm (inclusive); (c) applying an algorithm to thedetermined A1 and the determined A2 to generate a UCS, where thealgorithm includes a ratio of the determined A1 to the determined A2;and (d) characterizing a urine sample as a natural urine sample, aDNA-adulterated urine sample, a lyophilized urine sample, a dilutednatural urine sample, or a synthetic urine sample based on the UCS. Alsoprovided are methods of characterizing a urine sample from a subjectthat include: (a) determining the absorbance at a first wavelength (A1)and the absorbance at a second wavelength (A2) of a urine sample from asubject, where the A1 is from about 230 nm to about 250 nm (inclusive),and the A2 is from about 260 nm to about 340 nm (inclusive); (b)applying an algorithm to the determined A1 and the determined A2 togenerate a UCS, where the algorithm includes a ratio of the determinedA1 to the determined A2; and (c) characterizing a urine sample as anatural urine sample, a DNA-adulterated urine sample, a lyophilizedurine sample, a diluted natural urine sample, or a synthetic urinesample based on the UCS.

In some embodiments, the A1 is 230 nm, 231 nm, 232 nm, 233 nm, 234 nm,235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm,244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, or 250 nm. In someembodiments, the A1 is about 230 nm to about 250 nm, about 249 nm, about248 nm, about 247 nm, about 246 nm, about 245 nm, about 244 nm, about243 nm, about 242 nm, about 241 nm, about 240 nm, about 239 nm, about238 nm, about 237 nm, about 236 nm, about 235 nm, about 234 nm, about233 nm, or about 232 nm (inclusive); about 231 nm to about 250 nm, about249 nm, about 248 nm, about 247 nm, about 246 nm, about 245 nm, about244 nm, about 243 nm, about 242 nm, about 241 nm, about 240 nm, about239 nm, about 238 nm, about 237 nm, about 236 nm, about 235 nm, about234 nm, or about 233 nm (inclusive); about 232 nm to about 250 nm, about249 nm, about 248 nm, about 247 nm, about 246 nm, about 245 nm, about244 nm, about 243 nm, about 242 nm, about 241 nm, about 240 nm, about239 nm, about 238 nm, about 237 nm, about 236 nm, about 235 nm, or about234 nm (inclusive); about 233 nm to about 250 nm, about 249 nm, about248 nm, about 247 nm, about 246 nm, about 245 nm, about 244 nm, about243 nm, about 242 nm, about 241 nm, about 240 nm, about 239 nm, about238 nm, about 237 nm, about 236 nm, or about 235 nm (inclusive); about234 nm to about 250 nm, about 249 nm, about 248 nm, about 247 nm, about246 nm, about 245 nm, about 244 nm, about 243 nm, about 242 nm, about241 nm, about 240 nm, about 239 nm, about 238 nm, about 237 nm, or about236 nm (inclusive); about 235 nm to about 250 nm, about 249 nm, about248 nm, about 247 nm, about 246 nm, about 245 nm, about 244 nm, about243 nm, about 242 nm, about 241 nm, about 240 nm, about 239 nm, about238 nm, or about 237 nm (inclusive); about 236 nm to about 250 nm, about249 nm, about 248 nm, about 247 nm, about 246 nm, about 245 nm, about244 nm, about 243 nm, about 242 nm, about 241 nm, about 240 nm, about239 nm, or about 238 nm (inclusive); about 237 nm to about 250 nm, about249 nm, about 248 nm, about 247 nm, about 246 nm, about 245 nm, about244 nm, about 243 nm, about 242 nm, about 241 nm, about 240 nm, or about239 nm (inclusive); about 238 nm to about 250 nm, about 249 nm, about248 nm, about 247 nm, about 246 nm, about 245 nm, about 244 nm, about243 nm, about 242 nm, about 241 nm, or about 240 nm (inclusive); about239 nm to about 250 nm, about 249 nm, about 248 nm, about 247 nm, about246 nm, about 245 nm, about 244 nm, about 243 nm, about 242 nm, or about241 nm (inclusive); about 240 nm to about 250 nm, about 249 nm, about248 nm, about 247 nm, about 246 nm, about 245 nm, about 244 nm, about243 nm, or about 242 nm (inclusive); about 241 nm to about 250 nm, about249 nm, about 248 nm, about 247 nm, about 246 nm, about 245 nm, about244 nm, or about 243 nm (inclusive); about 242 nm to about 250 nm, about249 nm, about 248 nm, about 247 nm, about 246 nm, about 245 nm, or about244 nm (inclusive); about 243 nm to about 250 nm, about 249 nm, about248 nm, about 247 nm, about 246 nm, or about 245 nm (inclusive); about244 nm to about 250 nm, about 249 nm, about 248 nm, about 247 nm, orabout 246 nm (inclusive); about 245 nm to about 250 nm, about 249 nm,about 248 nm, or about 247 nm (inclusive); about 246 nm to about 250 nm,about 249 nm, or about 248 nm (inclusive); about 247 nm to about 250 nmor about 249 nm (inclusive); or about 248 nm to about 250 nm(inclusive).

In some embodiments of these methods, the A2 is 260 nm, 261 nm, 262 nm,263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm,272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm,281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm,290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm,299 nm, 300 nm, 301 nm, 302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm,308 nm, 309 nm, 310 nm, 311 nm, 312 nm, 313 nm, 314 nm, 315 nm, 316 nm,317 nm, 318 nm, 319 nm, 320 nm, 321 nm, 322 nm, 323 nm, 324 nm, 325 nm,326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm 334 nm,335 nm, 336 nm, 337 nm, 338 nm, 339 nm, or 340 nm. In some embodimentsof these methods, A2 is about 260 nm to about 340 nm, about 335 nm,about 330 nm, about 325 nm, about 320 nm, about 315 nm, about 310 nm,about 305 nm, about 300 nm, about 295 nm, about 290 nm, about 285 nm,about 280 nm, about 275 nm, about 270 nm, or about 265 nm (inclusive);about 265 nm to about 340 nm, about 335 nm, about 330 nm, about 325 nm,about 320 nm, about 315 nm, about 310 nm, about 305 nm, about 300 nm,about 295 nm, about 290 nm, about 285 nm, about 280 nm, about 275 nm, orabout 270 nm (inclusive); about 270 nm to about 340 nm, about 335 nm,about 330 nm, about 325 nm, about 320 nm, about 315 nm, about 310 nm,about 305 nm, about 300 nm, about 295 nm, about 290 nm, about 285 nm,about 280 nm, or about 275 nm (inclusive); about 275 nm to about 340 nm,about 335 nm, about 330 nm, about 325 nm, about 320 nm, about 315 nm,about 310 nm, about 305 nm, about 300 nm, about 295 nm, about 290 nm,about 285 nm, about 280 nm, about 275 nm, or about 270 nm (inclusive);about 270 nm to about 340 nm, about 335 nm, about 330 nm, about 325 nm,about 320 nm, about 315 nm, about 310 nm, about 305 nm, about 300 nm,about 295 nm, about 290 nm, about 285 nm, or about 280 nm (inclusive);about 280 nm to about 340 nm, about 335 nm, about 330 nm, about 325 nm,about 320 nm, about 315 nm, about 310 nm, about 305 nm, about 300 nm,about 295 nm, about 290 nm, about 285 nm, about 280 nm, about 275 nm, orabout 270 nm (inclusive); about 270 nm to about 340 nm, about 335 nm,about 330 nm, about 325 nm, about 320 nm, about 315 nm, about 310 nm,about 305 nm, about 300 nm, about 295 nm, about 290 nm, or about 285 nm(inclusive); about 285 nm to about 340 nm, about 335 nm, about 330 nm,about 325 nm, about 320 nm, about 315 nm, about 310 nm, about 305 nm,about 300 nm, about 295 nm, about 290 nm, about 285 nm, about 280 nm,about 275 nm, or about 270 nm (inclusive); about 270 nm to about 340 nm,about 335 nm, about 330 nm, about 325 nm, about 320 nm, about 315 nm,about 310 nm, about 305 nm, about 300 nm, about 295 nm, or about 290 nm(inclusive); about 290 nm to about 340 nm, about 335 nm, about 330 nm,about 325 nm, about 320 nm, about 315 nm, about 310 nm, about 305 nm,about 300 nm, or about 295 nm (inclusive); about 295 nm to about 340 nm,about 335 nm, about 330 nm, about 325 nm, about 320 nm, about 315 nm,about 310 nm, about 305 nm, or about 300 nm (inclusive); about 300 nm toabout 340 nm, about 335 nm, about 330 nm, about 325 nm, about 320 nm,about 315 nm, about 310 nm, or about 305 nm (inclusive); about 305 nm toabout 340 nm, about 335 nm, about 330 nm, about 325 nm, about 320 nm,about 315 nm, or about 310 nm (inclusive); about 310 nm to about 340 nm,about 335 nm, about 330 nm, about 325 nm, about 320 nm, or about 315 nm(inclusive); about 315 nm to about 340 nm, about 335 nm, about 330 nm,about 325 nm, or about 320 nm (inclusive); about 320 nm to about 340 nm,about 335 nm, about 330 nm, or about 325 nm (inclusive); about 325 nm toabout 340 nm, about 335 nm, or about 330 nm (inclusive); about 330 nm toabout 340 nm or about 335 nm (inclusive); or about 335 nm to about 340nm (inclusive). In some embodiments of any of these methods, the A1 is240 nm and the A2 is 280 nm.

In some embodiments of these methods, the UCS is generated using thefollowing formula:UCS=A1/A2.  (XI)

For Formula XI, if the determined UCS is below than a reference value(e.g., a threshold value), it indicates that the urine sample is asynthetic urine sample. An appropriate reference value (e.g., thresholdvalue) for identifying a synthetic urine sample can be determined by thetraining dataset (e.g., any of the exemplary training datasets describedherein). In some embodiments, a reference value (e.g., threshold value)for identifying a synthetic urine sample is selected because thereference value has the highest accuracy when the reference value isapplied to a training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, a reference value foridentifying a synthetic urine sample is selected because the referencevalue (e.g., threshold value) has the highest area under the curve (AUC)for receiver operating characteristic (ROC) when the reference value isapplied to a training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, the reference value(e.g., threshold value) can be any value from 0.01 to about 0.25, about0.24, about 0.23, about 0.22, about 0.21, about 0.20, about 0.19, about0.18, about 0.17, about 0.16, about 0.15, about 0.14, about 0.13, about0.12, about 0.11, about 0.10, about 0.09, about 0.08, about 0.07, about0.06, about 0.05, about 0.04, about 0.03, or about 0.02 (inclusive);about 0.02 to about 0.25, about 0.24, about 0.23, about 0.22, about0.21, about 0.20, about 0.19, about 0.18, about 0.17, about 0.16, about0.15, about 0.14, about 0.13, about 0.12, about 0.11, about 0.10, about0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, orabout 0.03 (inclusive); about 0.03 to about 0.25, about 0.24, about0.23, about 0.22, about 0.21, about 0.20, about 0.19, about 0.18, about0.17, about 0.16, about 0.15, about 0.14, about 0.13, about 0.12, about0.11, about 0.10, about 0.09, about 0.08, about 0.07, about 0.06, about0.05, or about 0.04 (inclusive); about 0.04 to about 0.25, about 0.24,about 0.23, about 0.22, about 0.21, about 0.20, about 0.19, about 0.18,about 0.17, about 0.16, about 0.15, about 0.14, about 0.13, about 0.12,about 0.11, about 0.10, about 0.09, about 0.08, about 0.07, about 0.06,or about 0.05 (inclusive); about 0.05 to about 0.25, about 0.24, about0.23, about 0.22, about 0.21, about 0.20, about 0.19, about 0.18, about0.17, about 0.16, about 0.15, about 0.14, about 0.13, about 0.12, about0.11, about 0.10, about 0.09, about 0.08, about 0.07, or about 0.06(inclusive); about 0.06 to about 0.25, about 0.24, about 0.23, about0.22, about 0.21, about 0.20, about 0.19, about 0.18, about 0.17, about0.16, about 0.15, about 0.14, about 0.13, about 0.12, about 0.11, about0.10, about 0.09, about 0.08, or about 0.07 (inclusive); about 0.07 toabout 0.25, about 0.24, about 0.23, about 0.22, about 0.21, about 0.20,about 0.19, about 0.18, about 0.17, about 0.16, about 0.15, about 0.14,about 0.13, about 0.12, about 0.11, about 0.10, about 0.09, or about0.08 (inclusive); about 0.08 to about 0.25, about 0.24, about 0.23,about 0.22, about 0.21, about 0.20, about 0.19, about 0.18, about 0.17,about 0.16, about 0.15, about 0.14, about 0.13, about 0.12, about 0.11,about 0.10, or about 0.09 (inclusive); about 0.09 to about 0.25, about0.24, about 0.23, about 0.22, about 0.21, about 0.20, about 0.19, about0.18, about 0.17, about 0.16, about 0.15, about 0.14, about 0.13, about0.12, about 0.11, or about 0.10 (inclusive); about 0.10 to about 0.25,about 0.24, about 0.23, about 0.22, about 0.21, about 0.20, about 0.19,about 0.18, about 0.17, about 0.16, about 0.15, about 0.14, about 0.13,about 0.12, or about 0.11 (inclusive); about 0.11 to about 0.25, about0.24, about 0.23, about 0.22, about 0.21, about 0.20, about 0.19, about0.18, about 0.17, about 0.16, about 0.15, about 0.14, about 0.13, orabout 0.12 (inclusive); about 0.12 to about 0.25, about 0.24, about0.23, about 0.22, about 0.21, about 0.20, about 0.19, about 0.18, about0.17, about 0.16, about 0.15, about 0.14, or about 0.13 (inclusive);about 0.13 to about 0.25, about 0.24, about 0.23, about 0.22, about0.21, about 0.20, about 0.19, about 0.18, about 0.17, about 0.16, about0.15, or about 0.14 (inclusive); about 0.14 to about 0.25, about 0.24,about 0.23, about 0.22, about 0.21, about 0.20, about 0.19, about 0.18,about 0.17, about 0.16, or about 0.15 (inclusive); about 0.15 to about0.25, about 0.24, about 0.23, about 0.22, about 0.21, about 0.20, about0.19, about 0.18, about 0.17, or about 0.16 (inclusive); about 0.16 toabout 0.25, about 0.24, about 0.23, about 0.22, about 0.21, about 0.20,about 0.19, about 0.18, or about 0.17 (inclusive); about 0.17 to about0.25, about 0.24, about 0.23, about 0.22, about 0.21, about 0.20, about0.19, or about 0.18 (inclusive); about 0.18 to about 0.25, about 0.24,about 0.23, about 0.22, about 0.21, about 0.20, or about 0.19(inclusive); about 0.19 to about 0.25, about 0.24, about 0.23, about0.22, about 0.21, or about 0.20 (inclusive); about 0.20 to about 0.25,about 0.24, about 0.23, about 0.22, or about 0.21 (inclusive); about0.21 to about 0.25, about 0.24, about 0.23, or about 0.22 (inclusive);about 0.22 to about 0.25, about 0.24, or about 0.23 (inclusive); about0.23 to about 0.25, or about 0.24 (inclusive); or about 0.24 to about0.25 (inclusive), when using Formula XI, and a UCS that is below thereference value indicates that the urine sample is a synthetic urinesample. In some embodiments, the reference value can be about 0.25,about 0.24, about 0.23, about 0.22, about 0.21, about 0.20, about 0.19,about 0.18, about 0.17, about 0.16, about 0.15, about 0.14, about 0.13,about 0.12, about 0.11, about 0.10, about 0.09, about 0.08, about 0.07,about 0.06, about 0.05, about 0.04, about 0.03, about 0.02, or about0.01, when using Formula XI, and a UCS that is below the reference valueindicates that the urine sample is a synthetic urine sample.

For Formula XI, if the determined UCS is above a reference value (e.g.,a threshold value), it indicates that the urine sample is a naturalurine sample. As described above, an appropriate reference value (e.g.,threshold value) for identifying a natural urine sample can bedetermined by the training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, a reference value(e.g., threshold value) for identifying a natural urine sample can beselected because the reference value has the highest accuracy when thereference value is applied to a training dataset (e.g., any of theexemplary training datasets described herein). In some embodiments, areference value for identifying a natural urine sample is selectedbecause the reference value (e.g., threshold value) has the highest areaunder the curve (AUC) for receiver operating characteristic (ROC) whenthe reference value is applied to a training dataset (e.g., any of theexemplary training datasets described herein). In some embodiments, thereference value (e.g., threshold value) can be any value from, e.g.,about 0.8 to about 2.0, about 1.95, about 1.9, about 1.85, about 1.8,about 1.75, about 1.7, about 1.65, about 1.6, about 1.55, about 1.5,about 1.45, about 1.4, about 1.35, about 1.3, about 1.25, about 1.2,about 1.15, about 1.1, about 1.05, about 1.0, about 0.95, about 0.9, orabout 0.85 (inclusive); about 0.85 to about 2.0, about 1.95, about 1.9,about 1.85, about 1.8, about 1.75, about 1.7, about 1.65, about 1.6,about 1.55, about 1.5, about 1.45, about 1.4, about 1.35, about 1.3,about 1.25, about 1.2, about 1.15, about 1.1, about 1.05, about 1.0,about 0.95, or about 0.9 (inclusive); about 0.9 to about 2.0, about1.95, about 1.9, about 1.85, about 1.8, about 1.75, about 1.7, about1.65, about 1.6, about 1.55, about 1.5, about 1.45, about 1.4, about1.35, about 1.3, about 1.25, about 1.2, about 1.15, about 1.1, about1.05, about 1.0, or about 0.95 (inclusive); about 0.95 to about 2.0,about 1.95, about 1.9, about 1.85, about 1.8, about 1.75, about 1.7,about 1.65, about 1.6, about 1.55, about 1.5, about 1.45, about 1.4,about 1.35, about 1.3, about 1.25, about 1.2, about 1.15, about 1.1,about 1.05, or about 1.0 (inclusive); about 1.0 to about 2.0, about1.95, about 1.9, about 1.85, about 1.8, about 1.75, about 1.7, about1.65, about 1.6, about 1.55, about 1.5, about 1.45, about 1.4, about1.35, about 1.3, about 1.25, about 1.2, about 1.15, about 1.1, or about1.05 (inclusive); about 1.05 to about 2.0, about 1.95, about 1.9, about1.85, about 1.8, about 1.75, about 1.7, about 1.65, about 1.6, about1.55, about 1.5, about 1.45, about 1.4, about 1.35, about 1.3, about1.25, about 1.2, about 1.15, or about 1.1 (inclusive); about 1.1 toabout 2.0, about 1.95, about 1.9, about 1.85, about 1.8, about 1.75,about 1.7, about 1.65, about 1.6, about 1.55, about 1.5, about 1.45,about 1.4, about 1.35, about 1.3, about 1.25, about 1.2, or about 1.15(inclusive); about 1.15 to about 2.0, about 1.95, about 1.9, about 1.85,about 1.8, about 1.75, about 1.7, about 1.65, about 1.6, about 1.55,about 1.5, about 1.45, about 1.4, about 1.35, about 1.3, about 1.25, orabout 1.2 (inclusive); about 1.2 to about 2.0, about 1.95, about 1.9,about 1.85, about 1.8, about 1.75, about 1.7, about 1.65, about 1.6,about 1.55, about 1.5, about 1.45, about 1.4, about 1.35, about 1.3, orabout 1.25 (inclusive); about 1.25 to about 2.0, about 1.95, about 1.9,about 1.85, about 1.8, about 1.75, about 1.7, about 1.65, about 1.6,about 1.55, about 1.5, about 1.45, about 1.4, about 1.35, or about 1.3(inclusive); about 1.3 to about 2.0, about 1.95, about 1.9, about 1.85,about 1.8, about 1.75, about 1.7, about 1.65, about 1.6, about 1.55,about 1.5, about 1.45, about 1.4, or about 1.35 (inclusive); about 1.35to about 2.0, about 1.95, about 1.9, about 1.85, about 1.8, about 1.75,about 1.7, about 1.65, about 1.6, about 1.55, about 1.5, about 1.45, orabout 1.4 (inclusive); about 1.4 to about 2.0, about 1.95, about 1.9,about 1.85, about 1.8, about 1.75, about 1.7, about 1.65, about 1.6,about 1.55, about 1.5, or about 1.45 (inclusive); about 1.45 to about2.0, about 1.95, about 1.9, about 1.85, about 1.8, about 1.75, about1.7, about 1.65, about 1.6, about 1.55, or about 1.5 (inclusive); about1.5 to about 2.0, about 1.95, about 1.9, about 1.85, about 1.8, about1.75, about 1.7, about 1.65, about 1.6, or about 1.55 (inclusive); about1.55 to about 2.0, about 1.95, about 1.9, about 1.85, about 1.8, about1.75, about 1.7, about 1.65, or about 1.6 (inclusive); about 1.6 toabout 2.0, about 1.95, about 1.9, about 1.85, about 1.8, about 1.75,about 1.7, or about 1.65 (inclusive); about 1.65 to about 2.0, about1.95, about 1.9, about 1.85, about 1.8, about 1.75, or about 1.7(inclusive); about 1.7 to about 2.0, about 1.95, about 1.9, about 1.85,about 1.8, or about 1.75 (inclusive); about 1.75 to about 2.0, about1.95, about 1.9, about 1.85, or about 1.8 (inclusive); about 1.8 toabout 2.0, about 1.95, about 1.9, or about 1.85 (inclusive); about 1.85to about 2.0, about 1.95, or about 1.9 (inclusive); about 1.9 to about2.0 or about 1.95 (inclusive); or about 1.95 to about 2.0 (inclusive),when using Formula XI, and a UCS that is above the reference valueindicates that the urine sample is a natural urine sample. In someembodiments, the reference value can be, e.g., about 0.80, about 0.85,about 0.90, about 0.95, about 1.0, about 1.05, about 1.1, about 1.15,about 1.2, about 1.25, about 1.3, about 1.35, about 1.4, about 1.45,about 1.5, about 1.55, about 1.6, about 1.65, about 1.7, about 1.75,about 1.8, about 1.85, about 1.9, about 1.95, or about 2.0, when usingFormula XI, and a UCS that is above the reference value indicates thatthe urine sample is a natural urine sample.

For Formula XI, if the determined UCS is within a range of referencevalues, it indicates that the urine sample is a diluted urine sample. Asdescribed above, a range of appropriate reference values for identifyinga diluted urine sample can be determined by the training dataset (e.g.,any of the exemplary training datasets described herein). In someembodiments, a range of reference values for identifying a diluted urinesample can be selected because the range of reference values has thehighest accuracy when the range of reference values is applied to atraining dataset (e.g., any of the exemplary training datasets describedherein). In some embodiments, a range of reference values foridentifying a diluted urine sample is selected because the range ofreference values has the highest area under the curve (AUC) for receiveroperating characteristic (ROC) when the range of reference values isapplied to a training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, the range of referencevalues (e.g., threshold value) can be, e.g., about 0.25 to about 0.80,about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about 0.68,about 0.66, about 0.64, about 0.62, about 0.60, about 0.58, about 0.56,about 0.54, about 0.52, about 0.50, about 0.48, about 0.46, about 0.44,about 0.42, about 0.40, about 0.38, about 0.36, about 0.34, about 0.32,about 0.30, about 0.28, or about 0.26 (inclusive); about 0.26 to about0.80, about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about0.68, about 0.66, about 0.64, about 0.62, about 0.60, about 0.58, about0.56, about 0.54, about 0.52, about 0.50, about 0.48, about 0.46, about0.44, about 0.42, about 0.40, about 0.38, about 0.36, about 0.34, about0.32, about 0.30, or about 0.28 (inclusive); about 0.28 to about 0.80,about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about 0.68,about 0.66, about 0.64, about 0.62, about 0.60, about 0.58, about 0.56,about 0.54, about 0.52, about 0.50, about 0.48, about 0.46, about 0.44,about 0.42, about 0.40, about 0.38, about 0.36, about 0.34, about 0.32,or about 0.30 (inclusive); about 0.30 to about 0.80, about 0.78, about0.76, about 0.74, about 0.72, about 0.70, about 0.68, about 0.66, about0.64, about 0.62, about 0.60, about 0.58, about 0.56, about 0.54, about0.52, about 0.50, about 0.48, about 0.46, about 0.44, about 0.42, about0.40, about 0.38, about 0.36, about 0.34, or about 0.32 (inclusive);about 0.32 to about 0.80, about 0.78, about 0.76, about 0.74, about0.72, about 0.70, about 0.68, about 0.66, about 0.64, about 0.62, about0.60, about 0.58, about 0.56, about 0.54, about 0.52, about 0.50, about0.48, about 0.46, about 0.44, about 0.42, about 0.40, about 0.38, about0.36, or about 0.34 (inclusive); about 0.34 to about 0.80, about 0.78,about 0.76, about 0.74, about 0.72, about 0.70, about 0.68, about 0.66,about 0.64, about 0.62, about 0.60, about 0.58, about 0.56, about 0.54,about 0.52, about 0.50, about 0.48, about 0.46, about 0.44, about 0.42,about 0.40, about 0.38, or about 0.36 (inclusive); about 0.36 to about0.80, about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about0.68, about 0.66, about 0.64, about 0.62, about 0.60, about 0.58, about0.56, about 0.54, about 0.52, about 0.50, about 0.48, about 0.46, about0.44, about 0.42, about 0.40, or about 0.38 (inclusive); about 0.38 toabout 0.80, about 0.78, about 0.76, about 0.74, about 0.72, about 0.70,about 0.68, about 0.66, about 0.64, about 0.62, about 0.60, about 0.58,about 0.56, about 0.54, about 0.52, about 0.50, about 0.48, about 0.46,about 0.44, about 0.42, or about 0.40 (inclusive); about 0.40 to about0.80, about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about0.68, about 0.66, about 0.64, about 0.62, about 0.60, about 0.58, about0.56, about 0.54, about 0.52, about 0.50, about 0.48, about 0.46, about0.44, or about 0.42 (inclusive); about 0.42 to about 0.80, about 0.78,about 0.76, about 0.74, about 0.72, about 0.70, about 0.68, about 0.66,about 0.64, about 0.62, about 0.60, about 0.58, about 0.56, about 0.54,about 0.52, about 0.50, about 0.48, about 0.46, or about 0.44(inclusive); about 0.44 to about 0.80, about 0.78, about 0.76, about0.74, about 0.72, about 0.70, about 0.68, about 0.66, about 0.64, about0.62, about 0.60, about 0.58, about 0.56, about 0.54, about 0.52, about0.50, about 0.48, or about 0.46 (inclusive); about 0.46 to about 0.80,about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about 0.68,about 0.66, about 0.64, about 0.62, about 0.60, about 0.58, about 0.56,about 0.54, about 0.52, about 0.50, or about 0.48 (inclusive); about0.48 to about 0.80, about 0.78, about 0.76, about 0.74, about 0.72,about 0.70, about 0.68, about 0.66, about 0.64, about 0.62, about 0.60,about 0.58, about 0.56, about 0.54, about 0.52, or about 0.50(inclusive); about 0.50 to about 0.80, about 0.78, about 0.76, about0.74, about 0.72, about 0.70, about 0.68, about 0.66, about 0.64, about0.62, about 0.60, about 0.58, about 0.56, about 0.54, or about 0.52(inclusive); about 0.52 to about 0.80, about 0.78, about 0.76, about0.74, about 0.72, about 0.70, about 0.68, about 0.66, about 0.64, about0.62, about 0.60, about 0.58, about 0.56, or about 0.54 (inclusive);about 0.54 to about 0.80, about 0.78, about 0.76, about 0.74, about0.72, about 0.70, about 0.68, about 0.66, about 0.64, about 0.62, about0.60, about 0.58, or about 0.56 (inclusive); about 0.56 to about 0.80,about 0.78, about 0.76, about 0.74, about 0.72, about 0.70, about 0.68,about 0.66, about 0.64, about 0.62, about 0.60, or about 0.58(inclusive); about 0.58 to about 0.80, about 0.78, about 0.76, about0.74, about 0.72, about 0.70, about 0.68, about 0.66, about 0.64, about0.62, or about 0.60 (inclusive); about 0.60 to about 0.80, about 0.78,about 0.76, about 0.74, about 0.72, about 0.70, about 0.68, about 0.66,about 0.64, or about 0.62 (inclusive); about 0.62 to about 0.80, about0.78, about 0.76, about 0.74, about 0.72, about 0.70, about 0.68, about0.66, or about 0.64 (inclusive); about 0.64 to about 0.80, about 0.78,about 0.76, about 0.74, about 0.72, about 0.70, about 0.68, or about0.66 (inclusive); about 0.66 to about 0.80, about 0.78, about 0.76,about 0.74, about 0.72, about 0.70, or about 0.68 (inclusive); about0.68 to about 0.80, about 0.78, about 0.76, about 0.74, about 0.72, orabout 0.70 (inclusive); about 0.70 to about 0.80, about 0.78, about0.76, about 0.74, or about 0.72 (inclusive); about 0.72 to about 0.80,about 0.78, about 0.76, or about 0.74 (inclusive); about 0.74 to about0.80, about 0.78, or about 0.76 (inclusive); about 0.76 to about 0.80 orabout 0.78 (inclusive); or about 0.78 to about 0.80 (inclusive), whenusing Formula XI, and a UCS that is within the range of the referencevalues indicates that the urine sample is a diluted urine sample.

In some embodiments, the UCS is generated using the following formula:UCS=10×log(A1/A2).  (XII)

For Formula XII, if the determined UCS is below than a reference value(e.g., a threshold value), it indicates that the urine sample is asynthetic urine sample. An appropriate reference value (e.g., thresholdvalue) for identifying a synthetic urine sample can be determined by thetraining dataset (e.g., any of the exemplary training datasets describedherein). In some embodiments, a reference value (e.g., threshold value)for identifying a synthetic urine sample is selected because thereference value has the highest accuracy when the reference value isapplied to a training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, a reference value foridentifying a synthetic urine sample is selected because the referencevalue (e.g., threshold value) has the highest area under the curve (AUC)for receiver operating characteristic (ROC) when the reference value isapplied to a training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, the reference value(e.g., threshold value) can be any value from, e.g., about −20.0 toabout −6.0, about −6.5, about −7.0, about −7.5, about −8.0, about −8.5,about −9.0, about −9.5, about −10.0, about −10.5, about −11.0, about−11.5, about −12.0, about −12.5, about −13.0, about −13.5, about −14.0,about −14.5, about −15.0, about −15.5, about −16.0, about −16.5, about−17.0, about −17.5, about −18.0, about −18.5, about −19.0, or about−19.5 (inclusive); about −19.5 to about −6.0, about −6.5, about −7.0,about −7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0,about −10.5, about −11.0, about −11.5, about −12.0, about −12.5, about−13.0, about −13.5, about −14.0, about −14.5, about −15.0, about −15.5,about −16.0, about −16.5, about −17.0, about −17.5, about −18.0, about−18.5, or about −19.0 (inclusive); about −19.0 to about −6.0, about−6.5, about −7.0, about −7.5, about −8.0, about −8.5, about −9.0, about−9.5, about −10.0, about −10.5, about −11.0, about −11.5, about −12.0,about −12.5, about −13.0, about −13.5, about −14.0, about −14.5, about−15.0, about −15.5, about −16.0, about −16.5, about −17.0, about −17.5,about −18.0, or about −18.5 (inclusive); about −18.5 to about −6.0,about −6.5, about −7.0, about −7.5, about −8.0, about −8.5, about −9.0,about −9.5, about −10.0, about −10.5, about −11.0, about −11.5, about−12.0, about −12.5, about −13.0, about −13.5, about −14.0, about −14.5,about −15.0, about −15.5, about −16.0, about −16.5, about −17.0, about−17.5, or about −18.0 (inclusive); about −18.0 to about −6.0, about−6.5, about −7.0, about −7.5, about −8.0, about −8.5, about −9.0, about−9.5, about −10.0, about −10.5, about −11.0, about −11.5, about −12.0,about −12.5, about −13.0, about −13.5, about −14.0, about −14.5, about−15.0, about −15.5, about −16.0, about −16.5, about −17.0, or about−17.5 (inclusive); about −17.5 to about −6.0, about −6.5, about −7.0,about −7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0,about −10.5, about −11.0, about −11.5, about −12.0, about −12.5, about−13.0, about −13.5, about −14.0, about −14.5, about −15.0, about −15.5,about −16.0, about −16.5, or about −17.0 (inclusive); about −17.0 toabout −6.0, about −6.5, about −7.0, about −7.5, about −8.0, about −8.5,about −9.0, about −9.5, about −10.0, about −10.5, about −11.0, about−11.5, about −12.0, about −12.5, about −13.0, about −13.5, about −14.0,about −14.5, about −15.0, about −15.5, about −16.0, or about −16.5(inclusive); about −16.5 to about −6.0, about −6.5, about −7.0, about−7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0, about−10.5, about −11.0, about −11.5, about −12.0, about −12.5, about −13.0,about −13.5, about −14.0, about −14.5, about −15.0, about −15.5, orabout −16.0 (inclusive); about −16.0 to about −6.0, about −6.5, about−7.0, about −7.5, about −8.0, about −8.5, about −9.0, about −9.5, about−10.0, about −10.5, about −11.0, about −11.5, about −12.0, about −12.5,about −13.0, about −13.5, about −14.0, about −14.5, about −15.0, orabout −15.5 (inclusive); about −15.5 to about −6.0, about −6.5, about−7.0, about −7.5, about −8.0, about −8.5, about −9.0, about −9.5, about−10.0, about −10.5, about −11.0, about −11.5, about −12.0, about −12.5,about −13.0, about −13.5, about −14.0, about −14.5, or about −15.0(inclusive); about −15.0 to about −6.0, about −6.5, about −7.0, about−7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0, about−10.5, about −11.0, about −11.5, about −12.0, about −12.5, about −13.0,about −13.5, about −14.0, or about −14.5 (inclusive); about −14.5 toabout −6.0, about −6.5, about −7.0, about −7.5, about −8.0, about −8.5,about −9.0, about −9.5, about −10.0, about −10.5, about −11.0, about−11.5, about −12.0, about −12.5, about −13.0, about −13.5, or about−14.0 (inclusive); about −14.0 to about −6.0, about −6.5, about −7.0,about −7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0,about −10.5, about −11.0, about −11.5, about −12.0, about −12.5, about−13.0, or about −13.5 (inclusive); about −13.5 to about −6.0, about−6.5, about −7.0, about −7.5, about −8.0, about −8.5, about −9.0, about−9.5, about −10.0, about −10.5, about −11.0, about −11.5, about −12.0,about −12.5, or about −13.0 (inclusive); about −13.0 to about −6.0,about −6.5, about −7.0, about −7.5, about −8.0, about −8.5, about −9.0,about −9.5, about −10.0, about −10.5, about −11.0, about −11.5, about−12.0, or about −12.5 (inclusive); about −12.5 to about −6.0, about−6.5, about −7.0, about −7.5, about −8.0, about −8.5, about −9.0, about−9.5, about −10.0, about −10.5, about −11.0, about −11.5, or about −12.0(inclusive); about −12.0 to about −6.0, about −6.5, about −7.0, about−7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0, about−10.5, about −11.0, or about −11.5 (inclusive); about −11.5 to about−6.0, about −6.5, about −7.0, about −7.5, about −8.0, about −8.5, about−9.0, about −9.5, about −10.0, about −10.5, or about −11.0 (inclusive);about −11.0 to about −6.0, about −6.5, about −7.0, about −7.5, about−8.0, about −8.5, about −9.0, about −9.5, about −10.0, or about −10.5(inclusive); about −10.5 to about −6.0, about −6.5, about −7.0, about−7.5, about −8.0, about −8.5, about −9.0, about −9.5, or about −10.0(inclusive); about −10.0 to about −6.0, about −6.5, about −7.0, about−7.5, about −8.0, about −8.5, about −9.0, or about −9.5 (inclusive);about −9.5 to about −6.0, about −6.5, about −7.0, about −7.5, about−8.0, about −8.5, or about −9.0 (inclusive); about −9.0 to about −6.0,about −6.5, about −7.0, about −7.5, about −8.0, or about −8.5(inclusive); about −8.5 to about −6.0, about −6.5, about −7.0, about−7.5, or about −8.0 (inclusive); about −8.0 to about −6.0, about −6.5,about −7.0, or about −7.5 (inclusive); about −7.5 to about −6.0, about−6.5, or about −7.0 (inclusive); about −7.0 to about −6.0, or about −6.5(inclusive); or about −6.5 to about −6.0 (inclusive), when using FormulaXII, and a UCS that is below the reference value indicates that theurine sample is a synthetic urine sample. In some embodiments, thereference value can be, e.g., about −6.0, about −6.5, about −7.0, about−7.5, about −8.0, about −8.5, about −9.0, about −9.5, about −10.0, about−10.5, about −11.0, about −11.5, about −12.0, about −12.5, about −13.0,about −13.5, about −14.0, about −14.5, about −15.0, about −15.5, about−16.0, about −16.5, about −17.0, about −17.5, about −18.0, about −18.5,about −19.0, about −19.5, or about −20.0, when using Formula XII, and aUCS that is below the reference value indicates that the urine sample isa synthetic urine sample.

For Formula XII, if the determined UCS is within a range of referencevalues, it indicates that the urine sample is a diluted urine sample. Asdescribed above, a range of appropriate reference values for identifyinga diluted urine sample can be determined by the training dataset (e.g.,any of the exemplary training datasets described herein). In someembodiments, a range of reference values for identifying a diluted urinesample can be selected because the range of reference values has thehighest accuracy when the range of reference values is applied to atraining dataset (e.g., any of the exemplary training datasets describedherein). In some embodiments, a range of reference values foridentifying a diluted urine sample is selected because the range ofreference values has the highest area under the curve (AUC) for receiveroperating characteristic (ROC) when the range of reference values isapplied to a training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, the range of referencevalues (e.g., threshold value) can be, e.g., about −6.0 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2,about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about −2.8,about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about −3.4,about −3.5, about −3.6, about −3.7, about −3.8, about −3.9, about −4.0,about −4.1, about −4.2, about −4.3, about −4.4, about −4.5, about −4.6,about −4.7, about −4.8, about −4.9, about −5.0, about −5.1, about −5.2,about −5.3, about −5.4, about −5.5, about −5.6, about −5.7, about −5.8,or about −5.9 (inclusive); about −5.9 to about −1.0, about −1.1, about−1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7, about−1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3, about−2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9, about−3.0, about −3.1, about −3.2, about −3.3, about −3.4, about −3.5, about−3.6, about −3.7, about −3.8, about −3.9, about −4.0, about −4.1, about−4.2, about −4.3, about −4.4, about −4.5, about −4.6, about −4.7, about−4.8, about −4.9, about −5.0, about −5.1, about −5.2, about −5.3, about−5.4, about −5.5, about −5.6, about −5.7, or about −5.8 (inclusive);about −5.8 to about −1.0, about −1.1, about −1.2, about −1.3, about−1.4, about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about−2.0, about −2.1, about −2.2, about −2.3, about −2.4, about −2.5, about−2.6, about −2.7, about −2.8, about −2.9, about −3.0, about −3.1, about−3.2, about −3.3, about −3.4, about −3.5, about −3.6, about −3.7, about−3.8, about −3.9, about −4.0, about −4.1, about −4.2, about −4.3, about−4.4, about −4.5, about −4.6, about −4.7, about −4.8, about −4.9, about−5.0, about −5.1, about −5.2, about −5.3, about −5.4, about −5.5, about−5.6, or about −5.7 (inclusive); about −5.7 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3,about −2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9,about −3.0, about −3.1, about −3.2, about −3.3, about −3.4, about −3.5,about −3.6, about −3.7, about −3.8, about −3.9, about −4.0, about −4.1,about −4.2, about −4.3, about −4.4, about −4.5, about −4.6, about −4.7,about −4.8, about −4.9, about −5.0, about −5.1, about −5.2, about −5.3,about −5.4, about −5.5, or about −5.6 (inclusive); about −5.6 to about−1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about−1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about−2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about−2.8, about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about−3.4, about −3.5, about −3.6, about −3.7, about −3.8, about −3.9, about−4.0, about −4.1, about −4.2, about −4.3, about −4.4, about −4.5, about−4.6, about −4.7, about −4.8, about −4.9, about −5.0, about −5.1, about−5.2, about −5.3, about −5.4, or about −5.5 (inclusive); about −5.5 toabout −1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5,about −1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1,about −2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7,about −2.8, about −2.9, about −3.0, about −3.1, about −3.2, about −3.3,about −3.4, about −3.5, about −3.6, about −3.7, about −3.8, about −3.9,about −4.0, about −4.1, about −4.2, about −4.3, about −4.4, about −4.5,about −4.6, about −4.7, about −4.8, about −4.9, about −5.0, about −5.1,about −5.2, about −5.3, or about −5.4 (inclusive); about −5.4 to about−1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about−1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about−2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about−2.8, about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about−3.4, about −3.5, about −3.6, about −3.7, about −3.8, about −3.9, about−4.0, about −4.1, about −4.2, about −4.3, about −4.4, about −4.5, about−4.6, about −4.7, about −4.8, about −4.9, about −5.0, about −5.1, about−5.2, or about −5.3 (inclusive); about −5.3 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3,about −2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9,about −3.0, about −3.1, about −3.2, about −3.3, about −3.4, about −3.5,about −3.6, about −3.7, about −3.8, about −3.9, about −4.0, about −4.1,about −4.2, about −4.3, about −4.4, about −4.5, about −4.6, about −4.7,about −4.8, about −4.9, about −5.0, about −5.1, or about −5.2(inclusive); about −5.2 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, about −2.3, about −2.4, about−2.5, about −2.6, about −2.7, about −2.8, about −2.9, about −3.0, about−3.1, about −3.2, about −3.3, about −3.4, about −3.5, about −3.6, about−3.7, about −3.8, about −3.9, about −4.0, about −4.1, about −4.2, about−4.3, about −4.4, about −4.5, about −4.6, about −4.7, about −4.8, about−4.9, about −5.0, or about −5.1 (inclusive); about −5.1 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2,about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about −2.8,about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about −3.4,about −3.5, about −3.6, about −3.7, about −3.8, about −3.9, about −4.0,about −4.1, about −4.2, about −4.3, about −4.4, about −4.5, about −4.6,about −4.7, about −4.8, about −4.9, or about −5.0 (inclusive); about−5.0 to about −1.0, about −1.1, about −1.2, about −1.3, about −1.4,about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about −2.0,about −2.1, about −2.2, about −2.3, about −2.4, about −2.5, about −2.6,about −2.7, about −2.8, about −2.9, about −3.0, about −3.1, about −3.2,about −3.3, about −3.4, about −3.5, about −3.6, about −3.7, about −3.8,about −3.9, about −4.0, about −4.1, about −4.2, about −4.3, about −4.4,about −4.5, about −4.6, about −4.7, about −4.8, or about −4.9(inclusive); about −4.9 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, about −2.3, about −2.4, about−2.5, about −2.6, about −2.7, about −2.8, about −2.9, about −3.0, about−3.1, about −3.2, about −3.3, about −3.4, about −3.5, about −3.6, about−3.7, about −3.8, about −3.9, about −4.0, about −4.1, about −4.2, about−4.3, about −4.4, about −4.5, about −4.6, about −4.7, or about −4.8(inclusive); about −4.8 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, about −2.3, about −2.4, about−2.5, about −2.6, about −2.7, about −2.8, about −2.9, about −3.0, about−3.1, about −3.2, about −3.3, about −3.4, about −3.5, about −3.6, about−3.7, about −3.8, about −3.9, about −4.0, about −4.1, about −4.2, about−4.3, about −4.4, about −4.5, about −4.6, or about −4.7 (inclusive);about −4.7 to about −1.0, about −1.1, about −1.2, about −1.3, about−1.4, about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about−2.0, about −2.1, about −2.2, about −2.3, about −2.4, about −2.5, about−2.6, about −2.7, about −2.8, about −2.9, about −3.0, about −3.1, about−3.2, about −3.3, about −3.4, about −3.5, about −3.6, about −3.7, about−3.8, about −3.9, about −4.0, about −4.1, about −4.2, about −4.3, about−4.4, about −4.5, or about −4.6 (inclusive); about −4.6 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2,about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about −2.8,about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about −3.4,about −3.5, about −3.6, about −3.7, about −3.8, about −3.9, about −4.0,about −4.1, about −4.2, about −4.3, about −4.4, or about −4.5(inclusive); about −4.5 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, about −2.3, about −2.4, about−2.5, about −2.6, about −2.7, about −2.8, about −2.9, about −3.0, about−3.1, about −3.2, about −3.3, about −3.4, about −3.5, about −3.6, about−3.7, about −3.8, about −3.9, about −4.0, about −4.1, about −4.2, about−4.3, or about −4.4 (inclusive); about −4.4 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3,about −2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9,about −3.0, about −3.1, about −3.2, about −3.3, about −3.4, about −3.5,about −3.6, about −3.7, about −3.8, about −3.9, about −4.0, about −4.1,about −4.2, or about −4.3 (inclusive); about −4.3 to about −1.0, about−1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about−1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about−2.3, about −2.4, about −2.5, about −2.6, about −2.7, about −2.8, about−2.9, about −3.0, about −3.1, about −3.2, about −3.3, about −3.4, about−3.5, about −3.6, about −3.7, about −3.8, about −3.9, about −4.0, about−4.1, or about −4.2 (inclusive); about −4.2 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3,about −2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9,about −3.0, about −3.1, about −3.2, about −3.3, about −3.4, about −3.5,about −3.6, about −3.7, about −3.8, about −3.9, about −4.0, or about−4.1 (inclusive); about −4.1 to about −1.0, about −1.1, about −1.2,about −1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8,about −1.9, about −2.0, about −2.1, about −2.2, about −2.3, about −2.4,about −2.5, about −2.6, about −2.7, about −2.8, about −2.9, about −3.0,about −3.1, about −3.2, about −3.3, about −3.4, about −3.5, about −3.6,about −3.7, about −3.8, about −3.9, or about −4.0 (inclusive); about−4.0 to about −1.0, about −1.1, about −1.2, about −1.3, about −1.4,about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about −2.0,about −2.1, about −2.2, about −2.3, about −2.4, about −2.5, about −2.6,about −2.7, about −2.8, about −2.9, about −3.0, about −3.1, about −3.2,about −3.3, about −3.4, about −3.5, about −3.6, about −3.7, about −3.8,or about −3.9 (inclusive); about −3.9 to about −1.0, about −1.1, about−1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7, about−1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3, about−2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9, about−3.0, about −3.1, about −3.2, about −3.3, about −3.4, about −3.5, about−3.6, about −3.7, or about −3.8 (inclusive); about −3.8 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2,about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about −2.8,about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about −3.4,about −3.5, about −3.6, or about −3.7 (inclusive); about −3.7 to about−1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about−1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about−2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about−2.8, about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about−3.4, about −3.5, or about −3.6 (inclusive); about −3.6 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2,about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about −2.8,about −2.9, about −3.0, about −3.1, about −3.2, about −3.3, about −3.4,or about −3.5 (inclusive); about −3.5 to about −1.0, about −1.1, about−1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7, about−1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3, about−2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9, about−3.0, about −3.1, about −3.2, about −3.3, or about −3.4 (inclusive);about −3.4 to about −1.0, about −1.1, about −1.2, about −1.3, about−1.4, about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about−2.0, about −2.1, about −2.2, about −2.3, about −2.4, about −2.5, about−2.6, about −2.7, about −2.8, about −2.9, about −3.0, about −3.1, about−3.2, or about −3.3 (inclusive); about −3.3 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3,about −2.4, about −2.5, about −2.6, about −2.7, about −2.8, about −2.9,about −3.0, about −3.1, or about −3.2 (inclusive); about −3.2 to about−1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about−1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about−2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about−2.8, about −2.9, about −3.0, or about −3.1 (inclusive); about −3.1 toabout −1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5,about −1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1,about −2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7,about −2.8, about −2.9, or about −3.0 (inclusive); about −3.0 to about−1.0, about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about−1.6, about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about−2.2, about −2.3, about −2.4, about −2.5, about −2.6, about −2.7, about−2.8, or about −2.9 (inclusive); about −2.9 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, about −1.9, about −2.0, about −2.1, about −2.2, about −2.3,about −2.4, about −2.5, about −2.6, about −2.7, or about −2.8(inclusive); about −2.8 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, about −2.3, about −2.4, about−2.5, about −2.6, or about −2.7 (inclusive); about −2.7 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, about −2.1, about −2.2,about −2.3, about −2.4, about −2.5, or about −2.6 (inclusive); about−2.6 to about −1.0, about −1.1, about −1.2, about −1.3, about −1.4,about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about −2.0,about −2.1, about −2.2, about −2.3, about −2.4, or about −2.5(inclusive); about −2.5 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, about −2.3, or about −2.4(inclusive); about −2.4 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, about −2.0, about −2.1, about −2.2, or about −2.3 (inclusive);about −2.3 to about −1.0, about −1.1, about −1.2, about −1.3, about−1.4, about −1.5, about −1.6, about −1.7, about −1.8, about −1.9, about−2.0, about −2.1, or about −2.2 (inclusive); about −2.2 to about −1.0,about −1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6,about −1.7, about −1.8, about −1.9, about −2.0, or about −2.1(inclusive); about −2.1 to about −1.0, about −1.1, about −1.2, about−1.3, about −1.4, about −1.5, about −1.6, about −1.7, about −1.8, about−1.9, or about −2.0 (inclusive); about −2.0 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about −1.7,about −1.8, or about −1.9 (inclusive); about −1.9 to about −1.0, about−1.1, about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, about−1.7, or about −1.8 (inclusive); about −1.8 to about −1.0, about −1.1,about −1.2, about −1.3, about −1.4, about −1.5, about −1.6, or about−1.7 (inclusive); about −1.7 to about −1.0, about −1.1, about −1.2,about −1.3, about −1.4, about −1.5, or about −1.6 (inclusive); about−1.6 to about −1.0, about −1.1, about −1.2, about −1.3, about −1.4, orabout −1.5 (inclusive); about −1.5 to about −1.0, about −1.1, about−1.2, about −1.3, or about −1.4 (inclusive); about −1.4 to about −1.0,about −1.1, about −1.2, or about −1.3 (inclusive); about −1.3 to about−1.0, about −1.1, or about −1.2 (inclusive); about −1.2 to about −1.0 orabout −1.1 (inclusive); or about −1.1 to about −1.0 (inclusive), whenusing Formula XII, and a UCS that is within the range of the referencevalues indicates that the urine sample is a diluted urine sample.

For Formula XII, if the determined UCS is above a reference value (e.g.,a threshold value), it indicates that the urine sample is a naturalurine sample. As described above, an appropriate reference value (e.g.,threshold value) for identifying a natural urine sample can bedetermined by the training dataset (e.g., any of the exemplary trainingdatasets described herein). In some embodiments, a reference value(e.g., threshold value) for identifying a natural urine sample can beselected because the reference value has the highest accuracy when thereference value is applied to a training dataset (e.g., any of theexemplary training datasets described herein). In some embodiments, areference value for identifying a natural urine sample is selectedbecause the reference value (e.g., threshold value) has the highest areaunder the curve (AUC) for receiver operating characteristic (ROC) whenthe reference value is applied to a training dataset (e.g., any of theexemplary training datasets described herein). In some embodiments, thereference value (e.g., threshold value) can be any value from, e.g.,about −1.0 to about 3.0, about 2.9, about 2.8, about 2.7, about 2.6,about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3,about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, about 0,about −0.1, about −0.2, about −0.3, about −0.4, about −0.5, about −0.6,about −0.7, about −0.8, or about −0.9 (inclusive); about −0.9 to about3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4,about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1,about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about0.4, about 0.3, about 0.2, about 0.1, about 0, about −0.1, about −0.2,about −0.3, about −0.4, about −0.5, about −0.6, about −0.7, or about−0.8 (inclusive); about −0.8 to about 3.0, about 2.9, about 2.8, about2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1,about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8,about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about0.1, about 0, about −0.1, about −0.2, about −0.3, about −0.4, about−0.5, about −0.6, or about −0.7 (inclusive); about −0.7 to about 3.0,about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4,about 0.3, about 0.2, about 0.1, about 0, about −0.1, about −0.2, about−0.3, about −0.4, about −0.5, or about −0.6 (inclusive); about −0.6 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5,about 0.4, about 0.3, about 0.2, about 0.1, about 0, about −0.1, about−0.2, about −0.3, about −0.4, or about −0.5 (inclusive); about −0.5 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5,about 0.4, about 0.3, about 0.2, about 0.1, about 0, about −0.1, about−0.2, about −0.3, or about −0.4 (inclusive); about −0.4 to about 3.0,about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4,about 0.3, about 0.2, about 0.1, about 0, about −0.1, about −0.2, orabout −0.3 (inclusive); about −0.3 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5,about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2,about 0.1, about 0, about −0.1, or about −0.2 (inclusive); about −0.2 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5,about 0.4, about 0.3, about 0.2, about 0.1, about 0, or about −0.1(inclusive); about −0.1 to about 3.0, about 2.9, about 2.8, about 2.7,about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4,about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1,or about 0 (inclusive); about 0 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5,about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2,or about 0.1 (inclusive); about 0.1 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5,about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, or about 0.2(inclusive); about 0.2 to about 3.0, about 2.9, about 2.8, about 2.7,about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4,about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about0.7, about 0.6, about 0.5, about 0.4, or about 0.3 (inclusive); about0.3 to about 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5,about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2,about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about0.5, or about 0.4 (inclusive); about 0.4 to about 3.0, about 2.9, about2.8, about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2,about 2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about1.5, about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9,about 0.8, about 0.7, about 0.6, or about 0.5 (inclusive); about 0.5 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about1.1, about 1.0, about 0.9, about 0.8, about 0.7, or about 0.6(inclusive); about 0.6 to about 3.0, about 2.9, about 2.8, about 2.7,about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4,about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, orabout 0.7 (inclusive); about 0.7 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5,about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, orabout 0.8 (inclusive); about 0.8 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5,about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, or about 0.9(inclusive); about 0.9 to about 3.0, about 2.9, about 2.8, about 2.7,about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4,about 1.3, about 1.2, about 1.1, or about 1.0 (inclusive); about 1.0 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, orabout 1.1 (inclusive); about 1.1 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5,about 1.4, about 1.3, or about 1.2 (inclusive); about 1.2 to about 3.0,about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, or about 1.3 (inclusive); about 1.3 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, about 1.5, or about 1.4 (inclusive); about 1.4 toabout 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, or about 1.5 (inclusive); about 1.5 to about 3.0,about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about 1.7,or about 1.6 (inclusive); about 1.6 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, about 1.9, about 1.8, or about 1.7 (inclusive); about1.7 to about 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5,about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, orabout 1.8 (inclusive); about 1.8 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about2.1, about 2.0, or about 1.9 (inclusive); about 1.9 to about 3.0, about2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about 2.3,about 2.2, about 2.1, or about 2.0 (inclusive); about 2.0 to about 3.0,about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about2.3, about 2.2, or about 2.1 (inclusive); about 2.1 to about 3.0, about2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about 2.3,or about 2.2 (inclusive); about 2.2 to about 3.0, about 2.9, about 2.8,about 2.7, about 2.6, about 2.5, about 2.4, or about 2.3 (inclusive);about 2.3 to about 3.0, about 2.9, about 2.8, about 2.7, about 2.6,about 2.5, or about 2.4 (inclusive); about 2.4 to about 3.0, about 2.9,about 2.8, about 2.7, about 2.6, or about 2.5 (inclusive); about 2.5 toabout 3.0, about 2.9, about 2.8, about 2.7, or about 2.6 (inclusive);about 2.6 to about 3.0, about 2.9, about 2.8, or about 2.7 (inclusive);about 2.7 to about 3.0, about 2.9, or about 2.8 (inclusive); about 2.8to about 3.0 or about 2.9 (inclusive); or about 2.9 to about 3.0(inclusive), when using Formula XI, and a UCS that is above thereference value indicates that the urine sample is a natural urinesample. In some embodiments, the reference value can be, e.g., about−1.0, about −0.9, about −0.8, about −0.7, about −0.6, about −0.5, about−0.4, about −0.3, about −0.2, about −0.1, about 0, about 0.1, about 0.2,about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5,about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8,about 2.9, or about 3.0, when using Formula XI, and a UCS that is abovethe reference value indicates that the urine sample is a natural urinesample.

Some examples of the methods described herein further include a step ofcomparing the determined absorbance at 280 nm of a urine sample to areference 280 nm absorbance value, where a decreased in the determinedabsorbance at 280 nm as compared to the reference 280 nm absorbancevalue indicates that the urine sample is a diluted urine sample. Areference 280 nm absorbance value can be, e.g., the absorbance at 280 nmof a control natural urine sample (e.g., a natural urine sample obtainedfrom a human subject not receiving one or more illegal or controlledsubstances). An average level of absorbance at 280 nm in control naturalurine samples can be obtained from a subject population (e.g., a subjectpopulation not receiving one or more illegal or controlled substances).A reference 280 nm absorbance value can be, e.g., an OD280 of about0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about0.40, about 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about0.70, about 0.75, about 0.80, about 0.85, about 0.90, about 0.95, about1.00, about 1.05, about 1.10, about 1.15, about 1.20, about 1.25, about1.30, about 1.35, about 1.40, about 1.45, about 1.50, about 1.55, about1.60, about 1.65, about 1.70, about 1.75, about 1.80, about 1.85, about1.90, about 1.95, or about 2.00. A reference 280 nm absorbance value canbe, e.g., about 0.10 to about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4,about 0.3, or about 0.2 (inclusive); about 0.2 to about 2.0, about 1.9,about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6,about 0.5, about 0.4, or about 0.3 (inclusive); about 0.3 to about 2.0,about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7,about 0.6, about 0.5, or about 0.4 (inclusive); about 0.4 to about 2.0,about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7,about 0.6, or about 0.5 (inclusive); about 0.5 to about 2.0, about 1.9,about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, or about 0.6(inclusive); about 0.6 to about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about1.0, about 0.9, about 0.8, or about 0.7 (inclusive); about 0.7 to about2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4,about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, or about 0.8(inclusive); about 0.8 to about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about1.0, or about 0.9 (inclusive); about 0.9 to about 2.0, about 1.9, about1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2,about 1.1, or about 1.0 (inclusive); about 1.0 to about 2.0, about 1.9,about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about1.2, or about 1.1 (inclusive); about 1.1 to about 2.0, about 1.9, about1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, or about 1.2(inclusive); about 1.2 to about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, or about 1.3 (inclusive); about 1.3 toabout 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, orabout 1.4 (inclusive); about 1.4 to about 2.0, about 1.9, about 1.8,about 1.7, about 1.6, or about 1.5 (inclusive); about 1.5 to about 2.0,about 1.9, about 1.8, about 1.7, or about 1.6 (inclusive); about 1.6 toabout 2.0, about 1.9, about 1.8, or about 1.7 (inclusive); about 1.7 toabout 2.0, about 1.9, or about 1.8 (inclusive); about 1.8 to about 2.0or about 1.9 (inclusive); or about 1.9 to about 2.0 (inclusive).

Some examples of the methods described herein further include a step ofcomparing the determined absorbance at 240 nm of a urine sample to areference 240 nm absorbance value, where a decreased in the determinedabsorbance at 240 nm as compared to the reference 240 nm absorbancevalue indicates that the urine sample is a diluted urine sample. Areference 240 nm absorbance value can be, e.g., the absorbance at 240 nmof a control natural urine sample (e.g., a natural urine sample obtainedfrom a human subject not receiving one or more illegal or controlledsubstances). An average level of absorbance at 240 nm in control naturalurine samples can be obtained from a subject population (e.g., a subjectpopulation not receiving one or more illegal or controlled substances).A reference 240 nm absorbance value can be, e.g., an OD240 of about 4.0,about 3.9, about 3.8, about 3.7, about 3.6, about 3.5, about 3.4, about3.3, about 3.2, about 3.1, about 3.0, about 2.9, about 2.8, about 2.7,about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4,about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about0.7, about 0.6, about 0.5, about 0.4, about 0.3, or about 0.2. Areference 240 nm absorbance value can be, e.g., about 0.2 to about 4.0,about 3.8, about 3.6, about 3.4, about 3.2, about 3.0, about 2.8, about2.6, about 2.4, about 2.2, about 2.0, about 1.8, about 1.6, about 1.4,about 1.2, about 1.0, about 0.8, about 0.6, or about 0.4 (inclusive);about 0.4 to about 4.0, about 3.8, about 3.6, about 3.4, about 3.2,about 3.0, about 2.8, about 2.6, about 2.4, about 2.2, about 2.0, about1.8, about 1.6, about 1.4, about 1.2, about 1.0, about 0.8, or about 0.6(inclusive); about 0.6 to about 4.0, about 3.8, about 3.6, about 3.4,about 3.2, about 3.0, about 2.8, about 2.6, about 2.4, about 2.2, about2.0, about 1.8, about 1.6, about 1.4, about 1.2, about 1.0, or about 0.8(inclusive); about 0.8 to about 4.0, about 3.8, about 3.6, about 3.4,about 3.2, about 3.0, about 2.8, about 2.6, about 2.4, about 2.2, about2.0, about 1.8, about 1.6, about 1.4, about 1.2, or about 1.0(inclusive); about 1.0 to about 4.0, about 3.8, about 3.6, about 3.4,about 3.2, about 3.0, about 2.8, about 2.6, about 2.4, about 2.2, about2.0, about 1.8, about 1.6, about 1.4, or about 1.2 (inclusive); about1.2 to about 4.0, about 3.8, about 3.6, about 3.4, about 3.2, about 3.0,about 2.8, about 2.6, about 2.4, about 2.2, about 2.0, about 1.8, about1.6, or about 1.4 (inclusive); about 1.4 to about 4.0, about 3.8, about3.6, about 3.4, about 3.2, about 3.0, about 2.8, about 2.6, about 2.4,about 2.2, about 2.0, about 1.8, or about 1.6 (inclusive); about 1.6 toabout 4.0, about 3.8, about 3.6, about 3.4, about 3.2, about 3.0, about2.8, about 2.6, about 2.4, about 2.2, about 2.0, or about 1.8(inclusive); about 1.8 to about 4.0, about 3.8, about 3.6, about 3.4,about 3.2, about 3.0, about 2.8, about 2.6, about 2.4, about 2.2, orabout 2.0 (inclusive); about 2.0 to about 4.0, about 3.8, about 3.6,about 3.4, about 3.2, about 3.0, about 2.8, about 2.6, about 2.4, orabout 2.2 (inclusive); about 2.2 to about 4.0, about 3.8, about 3.6,about 3.4, about 3.2, about 3.0, about 2.8, about 2.6, or about 2.4(inclusive); about 2.4 to about 4.0, about 3.8, about 3.6, about 3.4,about 3.2, about 3.0, about 2.8, or about 2.6 (inclusive); about 2.6 toabout 4.0, about 3.8, about 3.6, about 3.4, about 3.2, about 3.0, orabout 2.8 (inclusive); about 2.8 to about 4.0, about 3.8, about 3.6,about 3.4, about 3.2, or about 3.0 (inclusive); about 3.0 to about 4.0,about 3.8, about 3.6, about 3.4, or about 3.2 (inclusive); about 3.2 toabout 4.0, about 3.8, about 3.6, or about 3.4 (inclusive); about 3.4 toabout 4.0, about 3.8, or about 3.6 (inclusive); about 3.6 to about 4.0or about 3.8 (inclusive); or about 3.8 to about 4.0 (inclusive).

In some instances, a urine sample from a subject can be diluted prior toperforming the method. In such methods, a reference value to be used inthe methods can be generated from control urine samples (e.g., naturalurine samples and synthetic urine samples), where the control urinesamples are diluted to the same extent using the same diluent.

In some examples of any of these methods, the determining of theabsorbance at the A1 and the absorbance at the A2 of the urine sample isperformed using high-throughput processing.

The subject in these methods can be any of the subjects describedherein. The urine sample can be any of the urine samples or have any ofthe properties of urine samples described herein. Additional exemplaryaspects of these methods are described herein.

Methods of Characterizing a Urine Sample from a Subject that IncludeDetermining a UCI

Provided herein are methods of characterizing a urine sample from asubject. In some embodiments, the methods include (a) providing a urinesample from a subject; (b) determining one or more of the lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of the urine sample at two or more wavelengths oflight; (c) applying one or more eigenvectors derived from a principlecomponent algorithm to a standardized dataset derived from thedetermined light absorption, luminescence, phosphorescence,fluorescence, and chemiluminescence of the urine sample to generate aUrine Characterization Index (UCI) including one or more values forcorresponding principle component(s); and (d) characterizing a urinesample as a natural urine sample, a lyophilized urine sample, a dilutednatural urine sample, a synthetic urine sample, or achemically-adulterated urine sample based on the UCI. In someembodiments, the methods include: (a) providing a urine sample collectedfrom a subject; (b) determining the absorbance at a first wavelength(A1) and the absorbance at a second wavelength (A2) of the urine sample,where the A1 is from about 230 nm to about 250 nm (inclusive), and theA2 is from about 260 nm to about 230 nm (inclusive); (c) applying analgorithm to the determined A1 and the determined A2 to generate a UrineCharacterization Score (UCS), where the algorithm includes a ratio ofthe determined A1 to the determined A2; and (d) characterizing a urinesample as a natural urine sample, an adulterated urine sample, alyophilized urine sample, a diluted natural urine sample, or a syntheticurine sample based on the UCS.

In some embodiments of these methods, the light absorption of the urinesample at two or more (e.g., three, four, five, six, or seven)wavelengths of light is determined (e.g., any of the exemplarywavelengths of light described herein). In some embodiments of thesemethods, the light absorption of one or more (e.g., two, three, or four)of ultraviolet light wavelength(s), visible light wavelength(s),near-infrared light wavelength(s), and infrared light wavelength(s) isdetermined. In some embodiments of these methods, the light absorptionof the urine sample is determined at one or more wavelength(s) of about200 nm to about 1000 nm, about 980 nm, about 960 nm, about 940 nm, about920 nm, about 900 nm, about 880 nm, about 860 nm, about 840 nm, about820 nm, about 800 nm, about 780 nm, about 760 nm, about 740 nm, about720 nm, about 700 nm, about 680 nm, about 660 nm, about 640 nm, about620 nm, about 600 nm, about 580 nm, about 560 nm, about 540 nm, about520 nm, about 500 nm, about 480 nm, about 460 nm, about 440 nm, about420 nm, about 400 nm, about 380 nm, about 360 nm, about 340 nm, about320 nm, about 300 nm, about 295 nm, about 290 nm, about 285 nm, about280 nm, about 275 nm, about 270 nm, about 265 nm, about 260 nm, about255 nm, about 250 nm, about 245 nm, about 240 nm, about 235 nm, about230 nm, or about 225 nm (inclusive); about 225 nm to about 1000 nm,about 980 nm, about 960 nm, about 940 nm, about 920 nm, about 900 nm,about 880 nm, about 860 nm, about 840 nm, about 820 nm, about 800 nm,about 780 nm, about 760 nm, about 740 nm, about 720 nm, about 700 nm,about 680 nm, about 660 nm, about 640 nm, about 620 nm, about 600 nm,about 580 nm, about 560 nm, about 540 nm, about 520 nm, about 500 nm,about 480 nm, about 460 nm, about 440 nm, about 420 nm, about 400 nm,about 380 nm, about 360 nm, about 340 nm, about 320 nm, about 300 nm,about 295 nm, about 290 nm, about 285 nm, about 280 nm, about 275 nm,about 270 nm, about 265 nm, about 260 nm, about 255 nm, about 250 nm,about 245 nm, about 240 nm, about 235 nm, or about 230 nm (inclusive);about 230 nm to about 1000 nm, about 980 nm, about 960 nm, about 940 nm,about 920 nm, about 900 nm, about 880 nm, about 860 nm, about 840 nm,about 820 nm, about 800 nm, about 780 nm, about 760 nm, about 740 nm,about 720 nm, about 700 nm, about 680 nm, about 660 nm, about 640 nm,about 620 nm, about 600 nm, about 580 nm, about 560 nm, about 540 nm,about 520 nm, about 500 nm, about 480 nm, about 460 nm, about 440 nm,about 420 nm, about 400 nm, about 380 nm, about 360 nm, about 340 nm,about 320 nm, about 300 nm, about 295 nm, about 290 nm, about 285 nm,about 280 nm, about 275 nm, about 270 nm, about 265 nm, about 260 nm,about 255 nm, about 250 nm, about 245 nm, about 240 nm, or about 235 nm(inclusive); about 235 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm,about 340 nm, about 320 nm, about 300 nm, about 295 nm, about 290 nm,about 285 nm, about 280 nm, about 275 nm, about 270 nm, about 265 nm,about 260 nm, about 255 nm, about 250 nm, about 245 nm, or about 240 nm(inclusive); about 240 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm,about 340 nm, about 320 nm, about 300 nm, about 295 nm, about 290 nm,about 285 nm, about 280 nm, about 275 nm, about 270 nm, about 265 nm,about 260 nm, about 255 nm, about 250 nm, or about 245 nm (inclusive);about 245 nm to about 1000 nm, about 980 nm, about 960 nm, about 940 nm,about 920 nm, about 900 nm, about 880 nm, about 860 nm, about 840 nm,about 820 nm, about 800 nm, about 780 nm, about 760 nm, about 740 nm,about 720 nm, about 700 nm, about 680 nm, about 660 nm, about 640 nm,about 620 nm, about 600 nm, about 580 nm, about 560 nm, about 540 nm,about 520 nm, about 500 nm, about 480 nm, about 460 nm, about 440 nm,about 420 nm, about 400 nm, about 380 nm, about 360 nm, about 340 nm,about 320 nm, about 300 nm, about 295 nm, about 290 nm, about 285 nm,about 280 nm, about 275 nm, about 270 nm, about 265 nm, about 260 nm,about 255 nm, or about 250 nm (inclusive); about 250 nm to about 1000nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm, about 900nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm, about 800nm, about 780 nm, about 760 nm, about 740 nm, about 720 nm, about 700nm, about 680 nm, about 660 nm, about 640 nm, about 620 nm, about 600nm, about 580 nm, about 560 nm, about 540 nm, about 520 nm, about 500nm, about 480 nm, about 460 nm, about 440 nm, about 420 nm, about 400nm, about 380 nm, about 360 nm, about 340 nm, about 320 nm, about 300nm, about 295 nm, about 290 nm, about 285 nm, about 280 nm, about 275nm, about 270 nm, about 265 nm, about 260 nm, or about 255 nm(inclusive); about 255 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm,about 340 nm, about 320 nm, about 300 nm, about 295 nm, about 290 nm,about 285 nm, about 280 nm, about 275 nm, about 270 nm, about 265 nm, orabout 260 nm (inclusive); about 260 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm,about 460 nm, about 440 nm, about 420 nm, about 400 nm, about 380 nm,about 360 nm, about 340 nm, about 320 nm, about 300 nm, about 295 nm,about 290 nm, about 285 nm, about 280 nm, about 275 nm, about 270 nm, orabout 265 nm (inclusive); about 265 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm,about 460 nm, about 440 nm, about 420 nm, about 400 nm, about 380 nm,about 360 nm, about 340 nm, about 320 nm, about 300 nm, about 295 nm,about 290 nm, about 285 nm, about 280 nm, about 275 nm, or about 270 nm(inclusive); about 270 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm,about 340 nm, about 320 nm, about 300 nm, about 295 nm, about 290 nm,about 285 nm, about 280 nm, or about 275 nm (inclusive); about 275 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm,about 800 nm, about 780 nm, about 760 nm, about 740 nm, about 720 nm,about 700 nm, about 680 nm, about 660 nm, about 640 nm, about 620 nm,about 600 nm, about 580 nm, about 560 nm, about 540 nm, about 520 nm,about 500 nm, about 480 nm, about 460 nm, about 440 nm, about 420 nm,about 400 nm, about 380 nm, about 360 nm, about 340 nm, about 320 nm,about 300 nm, about 295 nm, about 290 nm, about 285 nm, or about 280 nm(inclusive); about 280 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm,about 340 nm, about 320 nm, about 300 nm, about 295 nm, about 290 nm, orabout 285 nm (inclusive); about 285 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm,about 460 nm, about 440 nm, about 420 nm, about 400 nm, about 380 nm,about 360 nm, about 340 nm, about 320 nm, about 300 nm, about 295 nm, orabout 290 nm (inclusive); about 290 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm,about 460 nm, about 440 nm, about 420 nm, about 400 nm, about 380 nm,about 360 nm, about 340 nm, about 320 nm, about 300 nm, or about 295 nm(inclusive); about 295 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm,about 340 nm, about 320 nm, or about 300 nm (inclusive); about 300 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm,about 800 nm, about 780 nm, about 760 nm, about 740 nm, about 720 nm,about 700 nm, about 680 nm, about 660 nm, about 640 nm, about 620 nm,about 600 nm, about 580 nm, about 560 nm, about 540 nm, about 520 nm,about 500 nm, about 480 nm, about 460 nm, about 440 nm, about 420 nm,about 400 nm, about 380 nm, about 360 nm, about 340 nm, or about 320 nm(inclusive); about 320 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, about 400 nm, about 380 nm, about 360 nm, orabout 340 nm (inclusive); about 340 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm,about 460 nm, about 440 nm, about 420 nm, about 400 nm, about 380 nm, orabout 360 nm (inclusive); about 360 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm,about 460 nm, about 440 nm, about 420 nm, about 400 nm, or about 380 nm(inclusive); about 380 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm,about 440 nm, about 420 nm, or about 400 nm (inclusive); about 400 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm,about 800 nm, about 780 nm, about 760 nm, about 740 nm, about 720 nm,about 700 nm, about 680 nm, about 660 nm, about 640 nm, about 620 nm,about 600 nm, about 580 nm, about 560 nm, about 540 nm, about 520 nm,about 500 nm, about 480 nm, about 460 nm, about 440 nm, or about 420 nm(inclusive); about 420 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, about 500 nm, about 480 nm, about 460 nm, orabout 440 nm (inclusive); about 440 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, about 480 nm, orabout 460 nm (inclusive); about 460 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm,about 560 nm, about 540 nm, about 520 nm, about 500 nm, or about 480 nm(inclusive); about 480 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm,about 540 nm, about 520 nm, or about 500 nm (inclusive); about 500 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm,about 800 nm, about 780 nm, about 760 nm, about 740 nm, about 720 nm,about 700 nm, about 680 nm, about 660 nm, about 640 nm, about 620 nm,about 600 nm, about 580 nm, about 560 nm, about 540 nm, or about 520 nm(inclusive); about 520 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, about 600 nm, about 580 nm, about 560 nm, orabout 540 nm (inclusive); about 540 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, about 580 nm, orabout 560 nm (inclusive); about 560 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm,about 660 nm, about 640 nm, about 620 nm, about 600 nm, or about 580 nm(inclusive); about 580 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm,about 640 nm, about 620 nm, or about 600 nm (inclusive); about 600 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm,about 800 nm, about 780 nm, about 760 nm, about 740 nm, about 720 nm,about 700 nm, about 680 nm, about 660 nm, about 640 nm, or about 620 nm(inclusive); about 620 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, about 700 nm, about 680 nm, about 660 nm, orabout 640 nm (inclusive); about 640 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, about 680 nm, orabout 660 nm (inclusive); about 660 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm,about 760 nm, about 740 nm, about 720 nm, about 700 nm, or about 680 nm(inclusive); about 680 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm,about 740 nm, about 720 nm, or about 700 nm (inclusive); about 700 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, about 820 nm,about 800 nm, about 780 nm, about 760 nm, about 740 nm, or about 720 nm(inclusive); about 720 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, about 800 nm, about 780 nm, about 760 nm, orabout 740 nm (inclusive); about 740 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, about 780 nm, orabout 760 nm (inclusive); about 760 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm,about 860 nm, about 840 nm, about 820 nm, about 800 nm, or about 780 nm(inclusive); about 780 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm,about 840 nm, about 820 nm, or about 800 nm (inclusive); about 800 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, about 920 nm,about 900 nm, about 880 nm, about 860 nm, about 840 nm, or about 820 nm(inclusive); about 820 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, about 900 nm, about 880 nm, about 860 nm, orabout 840 nm (inclusive); about 840 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, about 880 nm, orabout 860 nm (inclusive); about 860 nm to about 1000 nm, about 980 nm,about 960 nm, about 940 nm, about 920 nm, about 900 nm, or about 880 nm(inclusive); about 880 nm to about 1000 nm, about 980 nm, about 960 nm,about 940 nm, about 920 nm, or about 900 nm (inclusive); about 900 nm toabout 1000 nm, about 980 nm, about 960 nm, about 940 nm, or about 920 nm(inclusive); about 920 nm to about 1000 nm, about 980 nm, about 960 nm,or about 940 nm (inclusive); about 940 nm to about 1000 nm, about 980nm, or about 960 nm (inclusive); about 960 nm to about 1000 nm or about980 nm (inclusive); or about 980 nm to about 1000 nm (inclusive). Insome embodiments of these methods, the light absorption of the urinesample is determined at one or more wavelength(s) of about 100 nm toabout 180 nm (inclusive), about 100 nm to about 400 nm (inclusive),about 400 nm to about 700 nm (inclusive), about 700 nm to about 2500 nm(inclusive), and/or about 2500 nm to about 10,000 nm (inclusive). Insome embodiments of these methods, the light absorption of the urinesample is determined at one or more wavelength(s) selected from thegroup of: about 100 nm to about 180 nm (inclusive), about 180 nm toabout 400 nm (inclusive), about 400 nm to about 700 nm (inclusive),about 700 nm to about 2500 nm (inclusive), and about 2500 nm to 10000 nm(inclusive), or any combination thereof.

Some embodiments of these methods include determining one or more of theluminescence, phosphorescence, fluorescence, chemiluminescence, orbioluminescence of the urine sample. In some embodiments, the one ormore of the luminescence, phosphorescence, fluorescence,chemiluminescence, or bioluminecence of the urine sample is detected atany of the wavelengths or wavelength regions described herein.

Some embodiments of these methods can include, e.g., determining theemission phenomena of the urine sample (e.g., heat or black-bodyradiation). In some embodiments, the emission phenomena of the urinesample is detected at any of the wavelengths or wavelength regionsdescribed herein. Some embodiments of these methods include, e.g.,determining attenuated total reflectance and/or total internalreflectance of the urine sample.

In some embodiments of any of the methods, one or more of the lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of the urine sample arises following the applicationof electromagnetic energy to the urine sample and/or the introduction ofan agent (e.g., a chemical, a substrate, an enzyme, or an antibody) tothe urine sample.

Some embodiments of any of the methods described herein can include astep of applying a single eigenvector derived from a principle componentalgorithm to a standardized dataset derived from the determined lightabsorption, luminescence, phosphorescence, fluorescence, and/orchemiluminescence of the urine sample to generate a UrineCharacterization Index (UCI) including a value corresponding to oneprinciple component. Some embodiments of any of the methods describedherein can include a step of applying a single eigenvector derived froma principle component algorithm to a standardized dataset derived fromthe determined light absorption of the urine sample to generate a UrineCharacterization Index (UCI) including a value corresponding to oneprinciple component.

Some embodiments of any of the methods described herein can include astep of applying two eigenvectors derived from a principle componentalgorithm to a standardized dataset derived from the determined lightabsorption, luminescence, phosphorescence, fluorescence, and/orchemiluminescence of the urine sample to generate values intwo-dimensional principal component space. Some embodiments of any ofthe methods described herein can include a step of applying twoeigenvectors derived from a principle component algorithm to astandardized dataset derived from the determined light absorption of theurine sample to generate values in two-dimensional principal componentspace.

Some embodiments of any of the methods described herein can include astep of applying three eigenvectors derived from a principle componentalgorithm to a standardized dataset derived from the determined lightabsorption, luminescence, phosphorescence, fluorescence, and/orchemiluminescence of the urine sample to generate values inthree-dimensional principal component space. Some embodiments of any ofthe methods described herein can include a step of applying threeeigenvectors derived from a principle component algorithm to astandardized dataset derived from the determined light absorption of theurine sample to generate values in three-dimensional principal componentspace.

Some embodiments of any of the methods described herein can include astep of applying four eigenvectors derived from a principle componentalgorithm to a standardized dataset derived from the determined lightabsorption, luminescence, phosphorescence, fluorescence, and/orchemiluminescence of the urine sample to generate values infour-dimensional principal component space. Some embodiments of any ofthe methods described herein can include a step of applying foureigenvectors derived from a principle component algorithm to astandardized dataset derived from the determined light absorption of theurine sample to generate values in four-dimensional principal componentspace.

Some embodiments of any of these methods can further includecharacterizing a urine sample through the use of a clustering algorithm(e.g., a hierarchical clustering algorithm, a k-means clusteringalgorithm, or a statistical distribution model). Non-limiting examplesof clustering algorithms are described herein. Additional examples ofclustering algorithms are known in the art.

Some embodiments of any of these methods can further include furthercharacterizing a urine sample by performing regression analysis on thevalues of the one or more principle component(s). Some embodiments ofthese methods can further include comparing the determined UCI of theurine sample to a reference UCI of a natural urine sample, achemically-adulterated urine sample, a lyophilized urine sample, adiluted natural urine sample, or a synthetic urine sample. For example,a urine sample that has a determined UCI that falls within a specificrange of reference UCIs of natural urine samples can be identified as anatural urine sample; a urine sample that has a determined UCI thatfalls within a specific range of reference UCIs ofchemically-adulterated urine samples as a chemically-adulterated urinesample; a urine sample that has a determined UCI that falls within aspecific range of reference UCIs of lyophilized urine samples as alyophilized urine sample; a urine sample that has a determined UCI thatfalls within a specific range of reference UCIs of diluted natural urinesamples as a diluted natural urine sample; or a urine sample that has adetermined UCI that falls within a specific range of reference UCIs ofsynthetic urine samples as a synthetic urine sample. The reference UCIsfor natural urine samples, chemically-adulterated urine samples,lyophilized urine samples, diluted natural urine samples, and syntheticurine samples can be determined using a training dataset (e.g., any ofthe exemplary training datasets described herein).

The subject in these methods can be any of the subjects describedherein. The urine sample can be any of the urine samples or have any ofthe properties of urine samples described herein. Additional exemplaryaspects of these methods are described herein.

Spectrophotometers

The methods described herein include determining one or more of thelight absorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of the urine sample at two or more wavelengths oflight. The light absorption, luminescence, phosphorescence,fluorescence, and chemiluminescence of a urine sample can be determinedusing various spectrophotometers known in the art. Spectrophotometersthat can be used in any of the methods described herein are commerciallyavailable from a number of vendors, e.g., Beckman Coulter, Inc., AgilentTechnologies, Bibby Scientific Ltd., BioTek Instruments, BuckScientific, Cecil Instruments Ltd., Eppendorf North America, JASCO,Ocean Optics, Shimadzu, Terra Universal Inc., Thermo Scientific, andBiochrom. A high throughput UV-Vis spectrophotometer (e.g., UH4150UV-Visible-NIR Spectrophotometer from Hitachi High-Tech) can also beused in any of the methods described herein.

In some embodiments, a spectrophotometer can measure the lightabsorption of a urine sample at a wavelength from, e.g., about 200 nm toabout 2500 nm (inclusive), about 200 nm to about 1000 nm (inclusive),about 200 nm to about 340 nm (inclusive), about 230 nm to 250 nm(inclusive), about 260 nm to 340 nm (inclusive), about 200 nm to about300 nm (inclusive), about 300 nm to about 400 nm (inclusive), about 400nm to about 500 nm (inclusive), about 500 nm to about 600 nm(inclusive), about 600 nm to about 700 nm (inclusive), about 700 nm toabout 800 nm (inclusive), about 800 nm to about 900 nm (inclusive),and/or about 900 nm to about 1000 nm (inclusive).

Some examples of any of the methods described herein further include astep of experimentally diluting a urine sample from the subject (e.g.,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold,35-fold, 40-fold, 45-fold, 50-fold, 60-fold, or 64-fold dilution) priorto determination of the absorbance. As is known in the art, a urinesample that has an absorbance at 280 nm (or optionally an absorbance at240 nm) that exceeds an optical density of greater than 1.0, greaterthan 1.5, or greater than 2.0 may be diluted (e.g., in water) in orderto increase the sensitivity of the measurement of the absorbance by aspectrophotometer. In some embodiments, the concentration of a urinesample can be determined by ratiometric fluorescence indication.

Some embodiments of any of the methods described herein, further includea step of centrifuging a urine sample (or an aliquot of a urine sampleor an experimentally diluted natural urine sample) prior to determiningthe absorbance in order to remove any particulate matter (e.g.,mammalian cells, bacteria, nucleic acid, insoluble protein aggregates,soluble protein aggregates, and/or precipitated lipids). Methods forcentrifuging a sample (e.g., a urine sample) to remove particulatematerial are well-known in the art.

Training Dataset and Test Samples

The methods described herein further include creating a dataset fortraining samples (control urine samples, e.g., control natural urinesamples, control diluted natural urine samples, control lyophilizedurine samples, control chemically-adulterated urine samples, and/orcontrol synthetic urine samples) and one or more test samples (urinesamples from a subject). In some embodiments, a dataset includesmultiple data records, wherein each data record includes one or more ofthe light absorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of a urine sample at two or more wavelengths of light(e.g., as determined using a spectrophotometer).

In some embodiments, the dataset can include data derived from one ormore of the determined light absorption, luminescence, phosphorescence,fluorescence, and chemiluminescence of a urine sample at two or morewavelengths of light, e.g., the first derivative of the spectra, thesecond derivative of the spectra, the statistical moment, standardizedmoment, or standardized cumulant of a spectral feature including themean, variance, skewness, kurtosis otherwise known as the 1st, 2nd, 3rd,and 4th statistical moments, slope of the line tangent to any portion ofthe spectra, or results of the subtraction or addition of two or morespectra. In some embodiments, the dataset can include data indicatingthe area of one or more of the determined light absorption,luminescence, phosphorescence, fluorescence, and chemiluminescence of aurine sample at two or more wavelengths of light.

In some embodiments, a dataset includes one or more of the lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of a urine sample within a certain range ofwavelengths, for example, from about 200 nm to 340 nm (inclusive) (e.g.,including spectroscopic information at 200 nm, 210 nm, 220 nm, 230 nm,240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm,330 nm, and 340 nm). In some embodiments, a dataset can includespectroscopic information that is collected at every 1 nm, every 5 nm,every 10 nm, every 20 nm, every 50 nm, and every 100 nm wavelengthswithin a pre-determined wavelength region.

The methods described herein further include forming a training dataset.In some embodiments, samples for forming a training dataset include aplurality of natural urine samples, a plurality of synthetic urinesamples, a plurality of chemically-adulterated urine samples, aplurality of lyophilized urine samples, and/or a plurality of dilutednatural urine samples. In some embodiments, the number of urine samplesfor a particular type of urine samples should be chosen so as to achievea statistically meaningful result. In some embodiments, the trainingdatasets include about 10 to about 20 (inclusive) urine samples of eachtype of urine sample, about 10 to about 50 (inclusive) urine samples ofeach type of urine sample, about 50 to about 100 urine samples(inclusive) of each type of urine sample, about 100 to about 500(inclusive) or each type of urine sample, about 500 to about 1000 urinesamples (inclusive) of each type of urine sample, or more than 1000urine samples of each type of urine sample. In some embodiments, thetraining datasets include a total of greater than 50 urine samples,greater than 100 urine samples, greater than 150 urine samples, greaterthan 200 urine samples, greater than 250 urine samples, greater than 300urine samples, greater than 350 urine samples, greater than 400 urinesamples, greater than 450 urine samples, greater than 500 urine samples,greater than 600 urine samples, greater than 700 urine samples, greaterthan 800 urine samples, greater than 900 urine samples, or greater than1000 urine samples.

A test sample is a urine sample from a subject whose characteristicsneed to be determined. The present disclosure provides methods ofdetermining whether a test urine sample (a urine sample obtained from asubject) is a natural urine sample, a synthetic urine sample, achemically-adulterated urine sample (e.g., a surfactant-adulteratedurine sample, a lyophilized urine sample, or a diluted natural urinesample.

In some embodiments, one or more of the determined light absorption,luminescence, phosphorescence, fluorescence, and chemiluminescence attwo or more wavelengths of light for a test sample (a urine sampleobtained from a subject) and training samples are collected at the sametime, and the same experimental procedure is used. In some embodiments,one or more of the determined light absorption, luminescence,phosphorescence, fluorescence, and chemiluminescence at two or morewavelengths of light for the training samples is collected first and atraining dataset can be created prior to determining one or more of thedetermined light absorption, luminescence, phosphorescence,fluorescence, and chemiluminescence at two or more wavelengths of lightof a test sample (a urine sample from a subject).

Principal Component Analysis

Principal component analysis (PCA) is a statistical method that uses anorthogonal transformation to convert a set of observations of possiblycorrelated variables into a set of values of linearly uncorrelatedvariables called principal components. It finds the principal componentsof the dataset and transforms the data into a new, lower-dimensionalsubspace.

The transformation is defined in such a way that the first principalcomponent has the largest possible variance (that is, accounts for asmuch of the variability in the data as possible), and each succeedingprinciple component in turn has the highest variance possible under theconstraint that it is orthogonal to the preceding components. Theresulting vectors are an uncorrelated orthogonal basis set. PCA issensitive to the relative scaling of the original variables.

Mathematically, the principal components are the eigenvectors of thecovariance or correlation matrix of the original dataset. As thecovariance matrix or correlation matrix is symmetric, the eigenvectorsare orthogonal. The principal components (eigenvectors) correspond tothe direction (in the original n-dimensional space) with the greatestvariance in the data.

Each eigenvector has a corresponding eigenvalue. An eigenvalue is ascalar. The corresponding eigenvalue is a number that indicates how muchvariance there is in the data along that eigenvector (or principalcomponent). A large eigenvalue means that the principal componentindicates a large amount of the variance in the data. Similarly, aprincipal component with a very small eigenvalue indicates a smallamount variance in the data.

In some examples, the data are not normalized. In some examples, thedata are normalized. If the data are not normalized, attributes withlarge values and large variances (in absolute terms) will dominate thefirst principal component. Normalization transforms each attribute intomore or less to the same scale, so that each attribute has equalrepresentation during principal component analysis.

Non-limiting examples of how to perform PCA are described in, e.g.,Smith, Lindsay I, “A tutorial on principal components analysis.” CornellUniversity, USA 51, 2002, and Shlens, Jonathon, “A tutorial on principalcomponent analysis,” arXiv preprint arXiv: 1404.1100 (2014).

To apply principle component analysis in the disclosed methods, a set ofdata consisting of multiple measurements (e.g., one or more of the lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of a urine sample at two or more wavelengths of light)is created for each sample. The set of data for a sample can berepresented by a vector. For example, a vector X for Sample i can havemeasurements of light absorbance at m different wavelengths. Thus, eachvector is an m-dimensional vector, where m is the number of element.Each element can be a measurement. The elements in the vector can be themeasurements of the same type (e.g., light absorbance at a particularwavelength). They can be measurements of different types, e.g.,including both light absorbance and luminescence. In some embodiments,the vector can include elements that represent various characteristicsof the sample, e.g., spectroscopic information.

Within this description the index i will be used to denote thewavelength (range 1 to m) and the index j will be used to denote thesample within the data training set ranging from 1 to n. In order toestimate the population variance of spectral data about a specificwavelength, i, of a sample size of n, the statistical moments of eachmeasurement are calculated using, e.g., formula (I) and (II):μ_(i)=Σ_(j=1) ^(n) X _(ij) P _(ij)  (I)σ_(i) ²=Σ_(j=1) ^(n)(X _(ij)−μ_(i))² P _(ij)  (II)where μ_(i) is the mean, X_(ij) represents the measurement, e.g.,spectroscopic information obtained at the denoted wavelength i, for agiven sample and P_(ij) is the probability of X_(ij) at that i^(th)wavelength, for that sample. The variance at a specific wavelength,σ_(i) ² is calculated as the 2n^(d) central moment about the mean. Thei^(th) wavelength refers to a wavelength in a sequential index ofwavelengths beginning with the limits of the spectral range. Forexample, in some embodiments, if i=1, λ=200, the i+1 wavelength can be,e.g., 210 nm or 200.1 nm.

The result of Formula (I) and (II) for each wavelength can form twomatrices of dimensions 1×m: the mean and standard deviation arraysdenoted U and S, respectively, and contain the population varianceinformation for each type of measurement, e.g., light absorption atdifferent wavelengths as determined by a spectrophotometer.

X is defined as the array of spectral data (e.g. absorbance) at each ofm wavelengths for n specimens of the training set (that is, X is amatrix of dimension m×n, with elements X_(ij)). Let X′ denote thestandardized array of spectral data where the values are centered aboutthe corresponding means contained with U and divided by thecorresponding standard deviations contained within S. The correlationmatrix R is calculated by multiplying the transpose of X′ (that is,X′^(T)) with X′ and dividing by the degrees of freedom, n−1 (see, e.g.,Formula III).

$\begin{matrix}{R = \frac{X^{\prime\; T}X^{\prime}}{n - 1}} & ({III})\end{matrix}$

The spectral data matrix X is an i×j matrix with m number of wavelengthsand n number of spectral readings. For example, in one embodiment, i=15and corresponds to the wavelength between 200 nm and 340 nm, atintervals of 10 nm, and j=120, which is the number of samples in anexemplary training set for which there are spectral readings at eachwavelength. X′ denotes the standardized matrix in which each element isless than the mean and divided by the sample standard deviation.

The matrix W is a spectral data matrix where each element has hadsubtracted the corresponding element in matrix U. The covariance matrixC was calculated by multiplying the transpose of W (that is W^(T)) withW (see formula IV).

$\begin{matrix}{C = \frac{W^{T}W}{n - 1}} & ({IV})\end{matrix}$C captures the covariance between all possible pairs of measurements(e.g., light absorption at each of m wavelengths). The covariance valuesreflect the noise and redundancy in the measurements and is containedwith a matrix of dimensions m×m.

The eigenvalues¹, μ₁, for the correlation matrix are calculated and eacheigenvector, B_(i) (with dimension 1×n) for the correlation matrix isdetermined such that condition V is met:CB _(i)=λ_(i) B _(i)  (V)2 Note that here λ_(i) refers to the eigenvalue and not to aspectroscopic wavelength. However it is worth noting that for thisapplication these are the eigenvalues corresponding to wavelength rangesin the electromagnetic spectrum.

Each eigenvalue represents the amount of variance accounted for in thespectral data sets when normalized by the number of principal componentsin the data set (equal to the number of measurements). Let B be thematrix for which each column is composed of successive eigenvector(B_(i)) where i ranges from 1 to m. Given that each eigenvector is anm×1 matrix in which each element may also be considered as a regressioncoefficient for the principal components, B is an m×m matrix containingthe information for computation of the principal components of anelectromagnetic spectrum composed of n wavelength ranges.

The principal component matrix for each sample Y that can be calculatedby matrix multiplication of a matrix B transpose (that is B^(T)) withthe standardized spectral data X′:Y=B ^(T) X′  (VI)Thus each element of the principal component matrix Y, expressed asy_(ij), is a principal component for a given sample. In this work wehave shown that the first four (i=[1, . . . , 4]) principal componentscontain the majority (>90%) of the variance observed for a sample j.

Clustering Analysis

Some embodiments of any of the methods described herein, further includeperforming clustering analysis. Clustering is a procedure to groupsamples in such a way that samples in the same group (called a cluster)are more similar to each other than to those in other groups (clusters).Clustering algorithms include, but are not limited to, hierarchicalclustering algorithms, k-means clustering algorithms, and statisticaldistribution models.

Hierarchical clustering is a method of cluster analysis which seeks tobuild a hierarchy of clusters. The basic process of hierarchicalclustering can include:

(1) Starting by assigning each item to its own cluster, so that for Nitems, there will be N clusters. Each cluster initially contains justone item. Let the distances (similarities) between the clusters equalthe distances (similarities) between the items they contain.(2) Finding the closest (most similar) pair of clusters and merge theminto a single cluster.(3) Computing distances (similarities) between the new cluster and eachof the old clusters.(4) Repeating steps (2) and (3) until all items are clustered into asingle cluster of size N.

Detailed methods of clustering is described in numerous references. See,e.g., D'andrade, “U-Statistic Hierarchical Clustering” Psychometrika 4:58-67, 1978; Johnson, “Hierarchical Clustering Schemes” Psychometrika 2:241-254, 1967; and Borgatti, “How to explain hierarchical clustering,”Connections 17(2): 78-80, 1994.

In some embodiments, k-means clustering can be used to analyze resultsof principle component analysis. K-means clustering aims to partition nobservations into k clusters in which each observation belongs to thecluster with the nearest mean, serving as a prototype of the cluster.This results in a partitioning of the data space into Voronoi cells. Inone example, the algorithm proceeds as follows:

(1) Choosing k initial cluster centers (centroid), for example, choosingk observations at random.

(2) Computing point-to-cluster-centroid distances of all observations toeach centroid. There are many different ways to compute the distances,e.g., batch update, which assigns each observation to the cluster withthe closest centroid; and online update, which individually assignsobservations to a different centroid if the reassignment decreases thesum of the within-cluster, sum-of-squares point-to-cluster-centroiddistances.(3) Computing the average of the observations in each cluster to obtaink new centroid locations.(4) Repeating steps 2 through 3 until cluster assignments do not change,or the maximum number of iterations is reached.

An exemplary detailed method of implementing K-means clustering isdescribed, e.g., in U.S. Pat. No. 6,012,058; Kanungo et al., “Anefficient k-means clustering algorithm: Analysis and implementation,”IEEE transactions on pattern analysis and machine intelligence 24.7:881-892, 2002.

Furthermore, in some embodiments, clustering can be performed by astandard distribution model, e.g., multivariate normal distributionsused by the expectation-maximization (EM) algorithm. The EM iterationalternates between performing an expectation (E) step, which creates afunction for the expectation of the log-likelihood evaluated using thecurrent estimate for the parameters, and a maximization (M) step, whichcomputes parameters maximizing the expected log-likelihood found on theE step. These parameter-estimates are then used to determine thedistribution of the latent variables in the next E step. Exemplarymethods of implementing EM algorithms are described in, e.g., U.S. Pat.No. 6,615,205; WO 2011/162589; MacKay, “Information theory, inferenceand learning algorithms,” Cambridge University Press, 2003.

In some embodiments, the clustering analysis determines whether a urinesample from a subject is grouped or associated with natural urinesamples, synthetic urine samples, chemically-adulterated urine samples,lyophilized urine samples, or diluted natural urine samples, and thusdetermines whether a urine sample from a subject is a natural urinesample, a synthetic urine sample, a chemically-adulterated urine sample,a lyophilized urine sample, or a diluted natural urine sample.

Regression Analysis

Some embodiments of any of the methods described herein, further includeperforming regression analysis on one or more of the determined lightabsorption, luminescence, phosphorescence, fluorescence, andchemiluminescence of a urine sample at two or more wavelengths of light,or the data derived therefrom, e.g., results from principle componentanalysis.

A linear regression equation can be, e.g., expressed as:Y=α+β _(i) X ₁+β₂ X ₂+ . . . +β_(k) X _(k)+ε  (VII)Y, the dependent variable, indicates a quantitative measure, e.g., alikelihood score that the sample is a natural urine sample, a syntheticurine sample, a diluted natural urine sample, a lyophilized urinesample, or a chemically-adulterated urine sample. The dependent variableY depends on k explanatory variables (e.g., one or more of thedetermined light absorption, luminescence, phosphorescence,fluorescence, and chemiluminescence of a urine sample at two or morewavelengths of light, or results from principle component analysis),plus an error term that encompasses various unspecified omitted factors.In the above-identified model, the parameter β₁ gauges the effect of thefirst explanatory variable X₁ on the dependent variable Y. β₂ gives theeffect of the explanatory variable X₂ on Y.

A logistic regression model is a non-linear transformation of the linearregression. The logistic regression model is often referred to as the“logit” model and can be expressed as:ln[p/(1−p)]=α+β₁ X ₁+β₂ X ₂+ . . . +β_(k) X _(k)+ε  (VIII)

-   -   where:    -   α and ε are constants,    -   ln is the natural logarithm, log_((e)), where e=2.71828 . . . ,    -   p is the probability that the event Y occurs,    -   p/(1−p) is the “odds,” and    -   ln[p/(1−p)] is the log odds, or “logit.”

It will be appreciated by those of skill in the art that α and ε can befolded into a single constant, and expressed as α. In some embodiments,a single term α is used, and ε is omitted. The “logistic” distributionis an S-shaped distribution function. The logit distribution constrainsthe estimated probabilities (p) to lie between 0 and 1.

In one embodiment, the logistic regression model is expressed as:Y=α+Σβ _(i) X _(i),  (IX)where Y is a value indicating whether a test sample classifies with agroup of urine samples, e.g., natural urine samples, synthetic urinesamples, lyophilized urine samples, diluted natural urine samples, orchemically-adulterated urine samples, as opposed to another group ofurine samples. The probability that a test sample classifies with aparticular group of urine samples, as opposed to the other groups, canbe derived from Y. The higher the score, the higher the probability thatthe test sample classifies with the group of interest. Xi is anexplanatory variable. In some embodiments, Xi can be results obtainedfrom principle component analysis, e.g., X₁ can be the value(coordinate) for the first principle component, X₂ can be the value(coordinate) for the second principle component, and X₃ can be the value(coordinate) for the third principle component. In some embodiments, Xican be the one or more of the determined light absorption, luminescence,phosphorescence, fluorescence, and chemiluminescence of a urine sampleat two or more wavelengths of light, or data derived therefrom.

In some embodiments, the logistic regression model is fit by maximumlikelihood estimation (MLE). The coefficients (e.g., α, β1, β2, . . . )are determined by maximum likelihood. A likelihood is a conditionalprobability (e.g., P(Y|X), the probability of Y given X). The likelihoodfunction (L) measures the probability of observing the particular set ofdependent variable values (Y1, Y2, . . . , Yn) that occur in the dataset. In some embodiments, it is written as the product of theprobability of observing Y1, Y2, . . . , Yn:L=Prob(Y1,Y2, . . . ,Yn)=Prob(Y1)*Prob(Y2)* . . . Prob(Yn)  (X)

The higher the likelihood function, the higher the probability ofobserving the Ys in the sample. MLE involves finding the coefficients(α, β1, β2, . . . ) that make the log of the likelihood function (LL<0)as large as possible or −2 times the log of the likelihood function(−2LL) as small as possible. In MLE, some initial estimates of theparameters α, β1, β2, and so forth are made. Then, the likelihood of thedata given these parameter estimates is computed. The parameterestimates are improved, the likelihood of the data is recalculated. Thisprocess is repeated until the parameter estimates remain substantiallyunchanged (for example, a change of less than 0.01 or 0.001).Non-limiting examples of logistic regression and fitting logisticregression models are found in Hastie, “The Elements of StatisticalLearning,” Springer, N.Y., pp. 95-100, 2001.

Once the logistic regression equation coefficients and the logisticregression equation constant are determined, the model can be readilyapplied to a test subject to obtain Y. In one embodiment, Y can be usedto calculate probability (p) by solving the function Y=ln(p/(1−p)).

Platforms

The present disclosure also provides a platform for determining whethera urine sample comprises or consists essentially of, or is a naturalurine sample, a synthetic urine sample, a chemically-adulterated urinesample, a lyophilized urine sample, or a diluted natural urine sample.The platforms can include, e.g., a sample introduction mechanism, aspectrophotometric detection component, and an analysis system.

The sample introduction mechanism is designed for introducing a urinesample (e.g., a urine sample from a subject or a control urine sample ofany of the urine sample types described herein) for spectroscopicinterrogation and may include manual, automated, physical, or chemicalmanipulation of the sample. In some embodiments, the introduction of aurine sample includes manual introduction of urine sample (e.g., a urinesample from a subject or a control urine sample of any of the urinesample types described herein) by pipette or syringe, automatedintroduction of specimen by pipette or syringe using positivedisplacement, positive pressure, vacuum, suction, siphoning, capillaryaction, or electrophoresis.

A sample can be introduced into any suitable format for subsequentspectroscopic interrogation including chromatography vials, plastic(e.g., disposable cuvettes), glass cuvettes, quartz cuvettes, 8-wellstrips, 24-, 48-, 96-, or 384-well or other high-density format plateswith optically transparent windows for spectroscopic interrogation,sample containers for automated-analyzer systems, microscope slides,fluidic or microfluidic chambers comprised of polymer materials, glass,quartz, ceramic, stainless steel, or other metal alloys.

Physical manipulation of the samples during sample introduction caninclude dilution, pre-concentration, evaporation, lyophilization,sonication, ultrasonication, freezing, thawing, or any combinationthereof, to prepare the sample for spectroscopic interrogation.

Chemical manipulation of the samples during sample introduction caninclude flow-injection, liquid-liquid extraction (LLE), supported-liquidextraction (SLE), static headspace extraction, purge and trap,solid-phase extraction (SPE), solid-phase microextraction (SPME), sizeexclusion chromatography (SEC), protein precipitation, hydrophobicinteraction chromatography (HIC), thin layer chromatography (TLC), gaschromatography (GC), fast GC, two-dimensional gas chromatography(GC×GX), super-critical fluid chromatography (SFC), liquidchromatography, high-performance liquid chromatography (HPLC),ultra-high-performance liquid chromatography (UHPLC), two-dimensionalliquid chromatography (LC×LC), reverse-phase liquid chromatography(RPLC), normal-phase liquid chromatography, hydrophobic interactionliquid chromatography (HILIC), ion-pairing chromatography, capillaryzone electrophoresis (CZE), capillary isoelectric focusing, capillarygel electrophoresis, capillary electrokinetic chromatography, or anycombination thereof, prior to spectroscopic interrogation.

In some embodiments, a spectrophotometric detection component includes astand-alone device or instrument or can be fully-integrated into adevice, instrument, system, or platform. A spectrophotometric detectioncomponent can include a light source or combination of light sourceswhich emit across any portion or combination of the spectral regions asdescribed herein, a region where spectroscopic interrogation occurs or aregion of a device or instrument which performs any of the chemical orphysical manipulations of a urine sample, a spectroscopic analyzer whichmay be a prism, diffraction grating, or set of spectroscopic filtersincluding band-pass, band-stop, or notch filters, or an interferometer,a spectroscopic detector which may be either a phototude, photomulipliertube, microchannel plate, photographic film, charge-coupled device(CCD), charge-injection device, photoelectric detectors including, e.g.,CdZnTe, HgCdTe, photodiode, complementary metal-oxide-semiconductor(CMOS), a photovoltaic device, a thermal detector such as a bolometer,pyroelectric, cryogenic, or Golay detector, the necessary opticsincluding mirrors, lenses, and filters required to reflect, focus, orfilter wavelengths of light as they propogate from source through thesample, spectroscopic analyzer, and the detector.

The integrated device instrument or system may be of the followingtypes, a spectrophotometer, a spectroscope, a multi-well spectroscopicplate reader, a fluorimeter, a Fourier-transform infrared spectrometer,a UV-Vis spectrometer, a dispersive near-IR spectrometer, a VacuumUltraviolet Spectrometer, a diode-array detector, a CCD Camera, amicroscope, a fluorescence microscope, a microfluidic device-coupledspectrometer, a flow-cell detector, a GC detector, an LC-detector, anSFC-detector, an electrophoresis detector, or a hybrid detectionplatform that combines spectroscopic detection with mass spectrometry.

In some embodiments, the system or platform further includes, e.g., ananalysis system that performs a mathematical operation through a codedsoftware process which manipulates the raw spectroscopic information(e.g., one or more of the determined light absorption, luminescence,phosphorescence, fluorescence, and chemiluminescence at two or morewavelengths of light) to give a quantity which is related to thepresence of certain biological properties or proportional to theconcentration of chemical compounds in natural urine samples, and aredifferentiable from the quantities yielded by the same algorithm forsynthetic urine samples, lyophilized urine samples, diluted naturalurine samples, and chemical-adulterated urine samples.

In some embodiments, the methods described herein also includesperforming genetic analysis of genetic material present in the urinesample.

In some embodiments, the methods described herein also includesdetermining a chemical composition of a urine sample by massspectrometry analysis.

The result obtained by the methods described in this disclosure may becombined mathematically or qualitatively with the results of chemicaland physical validity testing including, the level of creatinine, thepH, the concentration of oxidizer(s), the specific gravity, the level ofnitrite, the level of glutaraldehyde, the concentration ofsurfactant(s), or temperature of the urine sample.

Analysis System and Computer Implementation

Any of the methods described herein can be, e.g., a computer-implementedmethod. In some embodiments, analysis as disclosed in the presentdisclosure can be implemented in digital electronic circuitry, intangibly-embodied computer software or firmware, in computer hardware,including the structures disclosed herein and their structuralequivalents, or in combinations of one or more thereof. Implementationsof the methods described herein can be implemented as one or morecomputer programs, i.e., one or more modules of computer programinstructions encoded on a tangible program carrier for execution by, orto control the operation of, a processing device. Alternatively or inaddition, the program instructions can be encoded on a propagated signalthat is an artificially generated signal, e.g., a machine-generatedelectrical, optical, or electromagnetic signal that is generated toencode information for transmission to suitable receiver apparatus forexecution by a processing device. A machine-readable medium can be,e.g., a machine-readable storage device, a machine-readable storagesubstrate, a random or serial access memory device, or a combination ofone or more thereof.

In some embodiments of any of the methods described herein, the analysismethods as disclosed herein are implemented in the form of computerprogram instructions and executed by a processing device. Suitableprogramming languages for expressing the program instructions include,but are not limited to, C, C++, Java, Python, SQL, Perl, Tcl/Tk,JavaScript, ADA, OCaml, Haskell, Scala, and statistical analysissoftware, such as SAS, R, MATLAB, SPSS, CORExpress® statistical analysissoftware, and Stata etc. Various aspects of the methods may be writtenin different computing languages from one another, and the variousaspects are caused to communicate with one another by appropriatesystem-level-tools available on a given system.

The processes and logic flows described in this specification can beperformed by one or more programmable computers executing one or morecomputer programs to perform functions by operating on input informationand generating output. The processes and logic flows can also beperformed by, and apparatus can also be implemented as, special purposelogic circuitry, e.g., an FPGA (field programmable gate array), an ASIC(application specific integrated circuit), or RISC.

Computers suitable for the execution of a computer program include, byway of example, general or special purpose microprocessors or both, orany other kind of central processing unit. Generally, a centralprocessing unit will receive instructions and information from aread-only memory or a random-access memory, or both. The essentialelements of a computer are a central processing unit for performing orexecuting instructions and one or more memory devices for storinginstructions and information. Generally, a computer will also include,or be operatively coupled to receive information from or transferinformation to, or both, one or more mass storage devices for storinginformation, e.g., magnetic, magneto optical disks, or optical disks.

Computer readable media suitable for storing computer programinstructions and information include various forms of non-volatilememory, media, and memory devices, including by way of examplesemiconductor memory devices, e.g., EPROM, EEPROM, and flash memorydevices; magnetic disks, e.g., internal hard disks or removable disks;magneto optical disks; and CD ROM and (Blue Ray) DVD-ROM disks. Theprocessor and the memory can be supplemented by, or incorporated in,special purpose logic circuitry.

To provide for interaction with a user, implementations of the methodsdescribed in this specification can be implemented on a computer havinga display device, e.g., a CRT (cathode ray tube) or LCD (liquid crystaldisplay) monitor, for displaying information to the user, a keyboard,and a pointing device, e.g., a mouse or a trackball, by which the usercan provide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well. In addition, a computer caninteract with a user by sending documents to and receiving documentsfrom a device that is used by the user; for example, by sending webpages to a web browser on a user's client device in response to requestsreceived from the web browser.

Implementations of the methods described in this specification can beimplemented in a computing system that includes a back end component,e.g., as an information server, or that includes a middleware component,e.g., an application server, or that includes a front end component,e.g., a client computer having a graphical user interface or a Webbrowser through which a user can interact with an implementation of thesubject matter described in this specification, or any combination ofone or more such back end, middleware, or front end components. Thecomponents of the system can be interconnected by any form or medium ofdigital information communication, e.g., a communication network.Examples of communication networks include a local area network (“LAN”)and a wide area network (“WAN”), e.g., the Internet.

The computing system can include clients and servers. A client andserver are generally remote from each other and typically interactthrough a communication network. The relationship of client and serverarises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other. In someembodiments, the server can be in the cloud via cloud computingservices.

While this specification includes many specific implementation details,these should not be construed as limitations on the scope of any of whatmay be claimed, but rather as descriptions of features that may bespecific to particular implementations. Certain features that aredescribed in this specification in the context of separateimplementations can also be implemented in combination in a singleimplementation. Conversely, various features that are described in thecontext of a single implementation can also be implemented in multipleimplementations separately or in any suitable sub-combination. Moreover,although features may be described above as acting in certaincombinations, one or more features from a combination can in some casesbe excised from the combination, and the claimed combination may bedirected to a sub-combination or variation of a sub-combination.

Obtaining an Additional Urine Sample

Some embodiments of any of the methods described herein can furtherinclude selecting a subject having a urine sample characterized as asynthetic urine sample, a diluted natural urine sample, achemically-adulterated urine sample, or a lyophilized urine sample, andobtaining an additional urine sample from the selected subject. In someembodiments, the additional urine sample is obtained through a witnessedurine test.

Some embodiments of any of the methods described herein can furtherinclude selecting a subject having a urine sample characterized as asynthetic urine sample, a diluted natural urine sample, achemically-adulterated urine sample, or a lyophilized urine sample, andobtaining a sample including blood, serum, hair, or plasma from theselected subject.

Assays to Determine the Level of One or More Drugs or Drug Metabolites

Some of the methods described herein further include performing an assayto determine the level of one or more (e.g., two, three, four, five,six, or seven) drugs and/or the level one or more (e.g., two, three,four, five, six, or seven) drug metabolites (e.g., any of the exemplarydrugs and/or drug metabolites described herein or known in the art) in asample (e.g., a urine sample identified using any of the methodsdescribed herein being a natural urine sample, an additional urinesample (e.g., any of the additional urine samples described herein), ora sample including blood, serum, hair, or plasma).

Non-limiting examples of drugs and drug metabolites include:Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabino-11-oic acid,11-hydroxy-Δ9-tetrahydrocannabinol,11-nor-9-carboxy-Δ9-tetrahydrocannabinol, ethyl glucuronide, ethylsulfate, morphine-3-glucuronide, morphine-6-glucu-ronide, amitriptyline,morphine 3,6-diglucuronide, morphine 3-ethereal sulfate, normorphine,cyclobenzaprine, norcodeine, codeine, normeperidine, norfentanyl,normorphine 6-glucoronide, 6-monoacetylmorphine, 6-monoacetylmorphine,3-monoacetylmorphine, buprenorphine, morphine, clobazam, hydromorphone,hydrocodone, norhydrocodone, oxymorphone, normethadol, methadol, EDDP,EMDP, benzoylecgonine, ecgonine methyl ester, norcocaine, carisoprodol,p-hydroxycocaine, m-hydroxycocaine, p-hydroxybenzoylecgonine,m-hydroxybenzoylecgonine, methamphetamine, meperidine, meprobamate,amphetamine, MDMA, MDEA, MDA, 5-(glutathion-S-yl)-alpha-methyldopamine,2,5-bis(glutathion-S-yl)-alpha-methyldopamine, free HMMA, DHMA sulfate,HMMA glucuronide, 7-aminoflunitrazepam, N-desmethylflunitrazepam,nitrazepam, N-desmethylclomipramine, N-desmethylcyclobenzaprine,doxepin, N-desmethylclobazam, desmethyldoxepin, 3-hydroxyflunitrazepam,gamma-hydroxybutyric acid, D-2-hydroxyglutaric acid, dehydronorketamine,maprotiline, imipramine, norketamine,4-phenyl-4-(1-piperidinyl)cyclohexanol, dextrorphan, N-acetyl mescaline,ortriptyline, desipramine, 10-OH-nortriptyline, nortriptyline, tramadol,O-desmethyl-cis-tramadol, desmethyl-nortriptyline, fentanyl,phenobarbital, amylobarbitone, 3′-hydroxyamylobarbitone, alpha-hydroxyalprazolam, zopiclone, zolpidem, 7-amino-clonazepam, 4-hydroxymidazolam,loprazolam, flurazepam, flurazepam, 7-aminoflunitrazepam, midazolam,1-hydroxymidazolam, norbuprenorphine, bromazepam, primidone,alpha-hydroxyalprazolam, 3-hydroxyflunitrazepam, estralozam,pentazocine, alprazolam, lorazepam, clonazepam, triazolam,desalkylfurazepam, flunitrazepam, propoxyphene, protriptyline, ritalinicacid, lormetazepam, alpha-hydroxytriazolam, desmethylflunitrazepam,methadone, diazepam, dothiepin, nordiazepam, oxazepam, methylphenidate,mianserin, naloxone, N-desmethylmirtazapine, mirtazapine,N-desmethyltapentadol, tapentadol, N-desmethyltrimipramine,trimipramine, metagynine, 7-hydroxymitragynine, AM2201, HU-210, JWH-018,JWH-018 5-pentanoic acid metabolite, JWH-073, JWH-073 4-butanoic acidmetabolite, JWH-073 N-(3-hydroxybutyl) metabolite, JWH-200, JWH-250,temazepam, marijuana, hashish, heroin, an opiate, cocaine, anamphetamine, phentermine, pregabalin, methamphetamine, a MDMA,flunitrazepam, GHB, ketamine, PCP, Salvia divinorum, dextromethorphan,dextromorphan, LSD, mescaline, psilocybin, mephedrone, methylone,3,4,-methylenedioxypyrovalerone (MDPV), an anabolic steroid, aninhalant, acetaminophen, hydrocodone, noroxycodone, oxycodone, tricyclicantidepressants, barbituates, and benzodiazepines.

A variety of urine drug assays and urine drug metabolite assays arecommercially available. For example, urine drug metabolite assays can bepurchased from American Screening Corp., Ameritox, Confirm Biosciences,Alibaba, Rapid Exams, and DrugConfirm.

An assay to determine the level of one or more drugs and/or the level ofone or more drug metabolites in a urine sample (e.g., any of the urinesamples described herein from any subject described herein) can beperformed at the same time as a method to characterize the urine sampleas a natural urine sample, a synthetic urine sample, a diluted naturalurine sample, a chemically-adulterated urine sample, or a lyophilizedurine sample (e.g., using any of the methods of characterizing a urinesample provided herein).

As is well known in the art, the determined level of the one or moredrugs and/or the determined level of the one or more drug metabolitescan be compared to reference values of the one or more drugs and/or theone or more drug metabolites (e.g., the level of the one or more drugsand/or the level of one or more drug metabolites in a subject that hasnot been administered a drug and/or an agent that is not metabolizedinto the one or more drug metabolites).

Some of the methods described herein also include methods of performinga principle component analysis on a natural urine sample containing adrug and/or a drug metabolite. As certain drugs or drug metabolites havecharacteristic spectra, the methods described in the present disclosure,e.g., principle component analysis, can also be used to distinguishnatural urine samples urine including or not including drugs and drugmetabolites.

Some embodiments of the methods where a subject is identified as havingan elevated level of one or more drug metabolites in the natural urinesample, the additional urine sample, or the sample including blood,serum, hair, or plasma, can further include admitting the subject into adrug dependency program, ceasing administration of the controlledsubstance to the subject, or reducing the dose or frequency ofadministration of the controlled substance to the subject. In someembodiments, the drug dependency program includes administering to thesubject a drug replacement therapy.

Additional Exemplary Steps

Some embodiments of any of the methods described herein can furtherinclude recording the characterization of the urine sample in asubject's medical record (e.g., a computer readable medium). Someembodiments of any of the methods described herein further includenotifying the subject's insurance provider, employer, or potentialfuture employer of the characterization of the urine sample. Someembodiments of any of the methods described herein further includingnotifying a pharmacist or a medical professional of the characterizationof the urine sample.

Some embodiments of any of the methods described herein can includeamplifying and sequencing nucleic acid present in the urine sample. Forexample, some embodiments of the methods further include: (a) providinga urine sample from a subject; (b) enriching the urine sample formammalian cells, if present; (c) isolating any genomic DNA from theenriched sample of step (b) to form an isolated genomic DNA test sample;(d) adding to the isolated genomic DNA test sample of step (c) a controlDNA to form a control sample or adding the control DNA to the enrichedsample of step (b) and then isolating DNA to form a control sample; (e)performing an assay to determine the presence of genomic DNA in theisolated genomic DNA sample of step (c) or the control sample of step(d); (f) performing an assay to determine the presence of the controlDNA in the control sample of step (d); and (g) identifying a urinesample having no detectable level of genomic DNA and having detectablecontrol DNA as including, consisting essentially or, or consisting ofsynthetic urine, or identifying a urine sample having a detectable levelof genomic DNA and having detectable control DNA as not including asynthetic urine. Non-limiting examples of the above steps are describedin U.S. Patent Application Publication No. 2016/0145684, incorporatedherein by reference in its entirety.

Some embodiments of the methods provided herein further include matchinga urine sample to a subject that include: (a) providing a urine samplefrom a subject; (b) enriching the urine sample for mammalian cells, ifpresent; (c) isolating any genomic DNA from the enriched sample of step(b) to form an isolated genomic DNA test sample; (d) adding to theisolated genomic DNA test sample of step (c) a control DNA to form acontrol sample or adding the control DNA to the enriched sample of step(b) and then isolating the DNA to form a control sample; (e) performingan assay to determine the genotype of at least 6 single nucleotidepolymorphisms (SNPs) in the isolated genomic DNA test sample of step (c)or the control sample of step (d); (f) comparing the genotype of the atleast 6 SNPs in the isolated genomic DNA test sample of step (c) or thecontrol sample of step (d) with the genotype of the at least 6 SNPs in acontrol cell sample (e.g., a buccal cell sample) from the subject; (g)performing an assay to determine the presence of the control DNA in thecontrol sample of step (d); and (h) identifying a urine sample having adetectable level of the control DNA and having the same genotype of theat least 6 SNPs in the isolated genomic DNA test sample of step (c) orthe control sample of step (d) as the genotype of the at least 6 SNPs inthe control cell sample as originating from the subject; or identifyinga urine sample having a detectable level of the control DNA and nothaving the same genotype of the at least 6 SNPs in the isolated genomicDNA test sample of step (c) or the control sample of step (d) as thegenotype of the at least 6 SNPs in the control cell sample as notoriginating from the subject. Non-limiting examples of the above stepsare described in U.S. Patent Application Publication No. 2016/0145684,incorporated herein by reference in its entirety.

Some embodiments of any of the methods described herein can furtherinclude detecting one or more of statherin, alpha-amylase, and lysozymein a urine sample. Statherin is a unique phoshoprotein found in saliva.Human statherin is 62 amino acids in length. The human statherin proteinsequence is shown below. A variety of antibodies that specifically bindto human statherin are commercially available (e.g., antibodiesavailable from Santa Cruz Biotech, Abcam, and Acris).

Human Statherin Protein (SEQ ID NO: 1)mkflvfafil almvsmigad sseekflrri grfgygygpy qpvpeqplyp qpyqpqyqqy tf

Human alpha-amylase is another protein that is present in saliva. Humanalpha-amylase is 511 amino acids. The human alpha-amylase proteinsequence is shown below. A variety of antibodies that specifically bindto human alpha-amylase are commercially available (e.g., antibodiesavailable from BioVision, AbCam, Sigma-Aldrich, Novus Biologicals, andNew England Biolabs).

Human Alpha-Amylase Protein (SEQ ID NO: 2)mkfflllfti gfcwaqyspn tqqgrtsivh lfewrwvdialecerylapk gfggvqvspp nenvaiynpf rpwweryqpvsyklctrsgn edefrnmvtr cnnvgvriyv davinhmcgnavsagtsstc gsyfnpgsrd fpavpysgwd fndgkcktgsgdienyndat qvrdcrltgl ldlalekdyv rskiaeymnhlidigvagfr ldaskhmwpg dikaildklh nlnsnwfpagskpfiyqevi dlggepikss dyfgngrvte fkygaklgtvirkwngekms ylknwgegwg fvpsdralvf vdnhdnqrghgaggasiltf wdarlykmav gfmlahpygf trvmssyrwprqfqngndvn dwvgppnnng vikevtinpd ttcgndwvcehrwrqirnmv ifrnvvdgqp ftnwydngsn qvafgrgnrgfivfnnddws fsltlqtglp agtycdvisg dkingnctgikiyvsddgka hfsisnsaed pfiaihaesk l

Human lysozyme is another protein that is present in saliva. Humanlysozyme is 148 amino acids. The human lysozyme protein sequence isshown below. A variety of antibodies that specifically bind to humanlysozyme are commercially available (e.g., antibodies available fromAbCam, Thermo Scientific, Novus Biologicals, and AbD Serotec).

Human Lysozyme (SEQ ID NO: 3)mkalivlglv llsvtvqgkv fercelartl krlgmdgyrgislanwmcla kwesgyntra tnynagdrst dygifqinsrywcndgktpg avnachlscs allqdniada vacakrvvrdpqgirawvaw rnrcqnrdvr qyvqgcgv

As is well-known in the art, a variety of antibody-based assays can beused to determine the presence of one or more of statherin,alpha-amylase, and lysozyme) in a urine sample. Non-limiting examples ofantibody-based assays include enzyme-linked immunosorbent assays,immunoblotting, protein chip, beads (e.g., magnetic beads) that arecoated with an antibody, immunoelectrophoresis, and immunoprecipitation.For example, any of the exemplary antibodies that bind specifically toone of statherin, alpha-amylase, or lysozyme can be used in any of theantibody-based assays described herein or known in the art to determinethe presence or level of statherin, alpha-amylase, or lysozyme in aurine sample.

Additional assays for determining the presence or level of one or morestatherin, alpha-amylase, and lysozyme in a urine sample are well knownin the art and include without limitation: mass spectrometry, enzymeactivity assays (e.g., using a detectable substrate or product),electrophoresis, and protein sequencing.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Methods of Characterizing a Urine Sample from a Subject

Experiments were performed to determine whether ultraviolet lightabsorbance of a urine sample can be used to determine if a urine sampleis a natural urine sample, a lyophilized urine sample, a diluted naturalurine sample, a synthetic urine sample, or a chemically-adulteratedurine sample.

The light absorption spectra of natural urine samples, lyophilized urinesamples, diluted natural urine samples, synthetic urine samples, andchemically-adulterated urine samples were obtained byspectrophotometers. FIG. 1 shows absorbance spectra of natural urinesample from humans. The absorbance characteristics can be describedqualitatively as strong absorbance throughout the ultraviolet region,particularly at wavelengths centered on 240, 280 and 300 nm. Withoutwishing to be bound by theory, it has been hypothesized that the strongultraviolet absorption characteristics are due to the presence of one ormore of: 1-aliphatic acyclic compounds, aliphatic heteromonocycliccompounds, aliphatic heteropolycyclic compounds, aliphatichomomonocyclic compounds, aliphatic homopolycyclic compounds, alkaloidsand derivatives, amino acids, peptides and analogues, aromaticheteromonocyclic compounds, aromatic heteropolycyclic compounds,aromatic homomonocyclic compounds, aromatic homopolycyclic compounds,carbohydrates and carbohydrate conjugates, homogenous metal compounds,homogenous non-metal compounds, inorganic compounds, lignans andnorlignans, lipids, mixed metal/non-metal compounds, nucleosides,nucleotides and analogs, organic acids and derivatives, organic halides,organometallic compounds, organophosphorous compounds, polyketides,tannins, bile acids, degradation or chemical-reaction, orenzymatic-reaction products of any of the above, or other compounds bothendogenous and xenobiotic in urine occurring due to metabolic processes.

While absorbance is directly proportional to concentration, the majorityof urine samples show a saturation effect due to the relatively highconcentration of chemical species that strongly absorb ultravioletlight.

FIG. 4 shows ultraviolet absorption spectra for lyophilized urinesamples, diluted natural urine samples, synthetic urine samples, andchemically-adulterated urine samples.

A dataset, containing the light absorption spectrum of these urinesamples, was created in order to establish and estimate of thepopulation variance. Each item in the training data set is a spectrumwhich contains absorbance at multiple wavelengths. In order to estimatethe population variance of a sample size of n, the statistical momentsof each wavelength were calculated using formula (I) and (II):μ_(i)=Σ_(j=1) ^(n) X _(ij) P _(ij)  (I)σ_(i) ²=Σ_(j=1) ^(n)(X _(ij)−μ_(i))² P _(ij)  (II)wherein μ_(i) is the mean, X_(ij) represents the measurement, e.g.,spectroscopic information obtained at the denoted wavelength i, for agiven sample and P_(ij) is the probability of X_(ij) at that i^(th)wavelength, for that sample. The variance at a specific wavelength,σ_(i) ² is calculated as the 2^(nd) central moment about the mean.

The result of Formula (I) and (II) for each wavelength formed twoarrays: the mean and standard deviation arrays denoted U and S forabsorbance at each wavelength.

X is defined as the array of spectral data (e.g. absorbance) at each ofm wavelengths for n specimens of the training set (that is, X is amatrix of dimension m×n, with elements X_(ij)). Let X′ denote thestandardized array of spectral data where the values are centered aboutthe corresponding means contained with U and divided by thecorresponding standard deviations contained within S. The correlationmatrix R is calculated by multiplying the transpose of X′ (that is,X′^(T)) with X′ and dividing by the degrees of freedom, n−1 (see, e.g.,Formula III).

$\begin{matrix}{R = \frac{X^{\prime\; T}X^{\prime}}{n - 1}} & ({III})\end{matrix}$

The spectral data matrix X is a i×j matrix with m number of wavelengthsand n number of spectral readings. For example, in one embodiment, wherei=15 and corresponds to the wavelength between 200 nm and 340 nm atintervals of 10 nm, and j=120, which is an exemplary number of samplesin the training set for which there are spectral readings at eachwavelength. X′ denotes the standardized matrix, in which each element isless than the mean and is divided by the sample standard deviation.

The matrix W is a spectral data matrix where each element has hadsubtracted the corresponding element in matrix U. The covariance matrixC was calculated by multiplying the transpose of W (that is W^(T)) withW (see formula IV).

$\begin{matrix}{C = \frac{W^{T}W}{n - 1}} & ({IV})\end{matrix}$C captures the covariance between all possible pairs of measurements(e.g., light absorption at each of n wavelengths). The covariance valuesreflect the noise and redundancy in the measurements and is containedwith a matrix of dimensions m×m.

The eigenvalues², λ_(i), for the correlation matrix are calculated andeach eigenvector, B_(i) (with dimension 1×m) for the correlation matrixis determined such that condition V is met:CB _(i)=λ_(i) B _(i)  (V)2 Note that here λ_(i) refers to the eigenvalue and not to aspectroscopic wavelength. However it is worth noting that for thisapplication these are the eigenvalues corresponding to wavelength rangesin the electromagnetic spectrum.

FIG. 3 is a Scree Plot indicating the % of total variance observed in atypical sample set per principal component (or eigenvalue). Theseresults indicate that 86% of the variance in the characteristic spectraof natural urine samples, lyophilized urine samples, diluted naturalurine samples, synthetic urine samples, and chemically-adulterated urinesamples can be accounted for by the first three principle components.

Table 1 shows the extent to which each wavelength range contributes to aspecific principal component. For the tested urine samples, principalcomponent 1 correlates with the variance observed between 260 and 300nm, where as principal component 2 is composed chiefly of the varianceat 230-250 nm. Thus, the first two principal components correlate oranti-correlate with wavelengths centered at 280-300 nm and 240 nm. Thisexplains why simpler algorithms based upon the ratio or relativeabsorbance at 240 nm and 280 nm can be useful.

TABLE 1 The eigenvalues for the first 4 principal components are shown(2nd row, grey box). The eigenvectors for the correlation matrix showwhich of the principal components give rise to the most variance in eachspectral region. Eigenvectors with absolute value > 0.3 have beenhighlighted to indicate the relationships. λ ϵ₁ ϵ₂ ϵ₃ ϵ₄ (nm)8.526359185 3.010215805 1.324644831 0.994120112 200 0.034623586−0.159491402 0.321332164 0.858766315 210 0.11690475 0.1700904360.608180694 −0.108715591 220 −0.123168703 0.227883361 0.604015091−0.248533682 230 0.013949483 0.551478495 −0.19076237 0.1572497 2400.012596323 0.551082751 −0.191034495 0.160662149 250 −0.200507240.435270894 0.140259216 −0.045323324 260 −0.314971054 0.1245059690.06743741 0.235687282 270 −0.318209273 0.00130829 −0.0563443780.183002047 280 −0.328638531 −0.044062678 −0.010629273 0.116787428 290−0.324398063 −0.101867851 0.084267126 0.023132726 300 −0.305410201−0.174520286 0.140254063 −0.080321726 310 −0.315927261 −0.1759454490.027782547 −0.129656334 320 −0.328297643 −0.021809147 −0.088052023−0.083989453 330 −0.328674789 0.049497719 −0.11421298 −0.042247557 340−0.326824678 0.065250359 −0.115296519 −0.032003884

The principal component matrix for each sample Y that can be calculatedby matrix multiplication of a matrix B transpose (that is B^(T)) withthe standardized spectral data X′:Y=B ^(T) X′  (VI)

Thus each element of the principal component matrix Y, expressed asy_(ij), is a principal component for a given sample. In this work wehave shown that the first four (i=[1, . . . , 4]) principal componentscontain the majority (>90%) of the variance observed for a sample j.

Using the first 4 principal components, a reduced data model of thespectral information was reconstructed for each urine sample. FIGS. 2and 5 show how well the original raw data (the raw data from FIGS. 1 and4, respectively) can be represented by a subset of principal components.As expected, the reduced data model captures the key spectral featuresof the original data (compare FIGS. 2 and 5 to FIGS. 1 and 4,respectively).

A principal component cluster plot was constructed using the first threeprincipal components. FIG. 6 shows a cluster plot for lyophilized urinesamples, diluted natural urine samples, synthetic urine samples, andchemically-adulterated urine samples based on the three principlecomponents. Statistical analysis of these clusters were used todifferentiate urine samples into different categories.

Example 2 Characterizing Synthetic and Chemically-Adulterated UrineSamples from a Subject

As shown in Example 1, principal components 1 and 2 account for 75% ofthe sample variance, and the first principal component correlates withthe variance observed between 260 nm and 300 nm, and the secondprincipal component correlates with the variance observed between 230 nmand 250 nm. Thus, new algorithms were developed to differentiatesynthetic urine from a natural urine sample by determining therelationship between absorbance at a wavelength between 260 and 300 nmand the absorbance at a wavelength between 230 and 250 nm.

Absorbance at 240 nm and 280 nm are determined, and Formulas (XIII) and(XIV) are applied to these data.

$\begin{matrix}{{{SynScore}\mspace{14mu} 1} = \frac{\lambda_{240}}{\lambda_{280}}} & ({XIII}) \\{{{SynScore}\mspace{14mu} 2} = {10 \times {\log\left( \frac{\lambda_{240}}{\lambda_{280}} \right)}}} & ({XIV})\end{matrix}$

If SynScore 1 is greater than 3, it indicates that the sample may be asynthetic urine sample due to low UV absorbance at 280 nm.

SynScore 2 is based on the logarithm of the absorbance ratio. SynScore 2ranges from approximately 0 to about 50. For SynScore 2, a value greaterthan 5 indicates that sample is a synthetic urine sample. SynScore 2 fora natural urine sample ranges from 0 to 1.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of characterizing a urine sample from a subject, the method comprising: (a) providing a urine sample from a subject; (b) determining one or more of the light absorption, luminescence, phosphorescence, fluorescence, and chemiluminescence of the urine sample at two or more wavelengths of light; (c) applying one or more eigenvectors derived from a principle component algorithm to a standardized dataset derived from the determined light absorption, luminescence, phosphorescence, fluorescence, and chemiluminescence of the urine sample to generate a Urine Characterization Index (UCI) comprising one or more values for corresponding principle component(s); and (d) characterizing a urine sample as a natural urine sample, a lyophilized urine sample, a diluted natural urine sample, a synthetic urine sample, or a chemically-adulterated urine sample based on the UCI.
 2. The method of claim 1, wherein step (b) comprises determining the light absorption of the urine sample.
 3. The method of claim 2, wherein step (b) comprises determining the light absorption of one or more of ultraviolet light wavelength(s), visible light wavelength(s), near-infrared light wavelength(s), and infrared absorption light wavelengths.
 4. The method of claim 2, wherein step (b) comprises determining the light absorption of the urine sample at wavelengths of about 200 nm to about 1000 nm.
 5. The method of claim 2, wherein step (c) comprises applying an eigenvector derived from the principle component algorithm to a dataset derived from the determined light absorption to generate a value for one principle component.
 6. The method of claim 1, wherein step (c) comprises applying two eigenvectors derived from the principle component algorithm to a dataset derived from the determined light absorption, luminescence, phosphorescence, fluorescence, chemiluminescence, or bioluminescence to generate values in two-dimensional principle component space.
 7. The method of claim 1, wherein step (c) comprising applying three eigenvectors derived from the principle component algorithm to a dataset derived from the determined light absorption, luminescence, phosphorescence, fluorescence, chemiluminescence, or bioluminescence to generate values in three-dimensional principle component space.
 8. The method of claim 1, wherein step (c) comprising applying four eigenvectors derived from the principle component algorithm to a dataset derived from the determined light absorption, luminescence, phosphorescence, fluorescence, chemiluminescence, or bioluminescence to generate values in four-dimensional principle component space.
 9. The method of claim 1, wherein step (d) comprises characterizing a urine sample through the use of a clustering algorithm.
 10. The method of claim 1, wherein step (d) comprises characterizing a urine sample by performing regression analysis on the values of principle components.
 11. The method of claim 1, wherein step (d) comprises comparing the UCI of the sample to a UCI of a natural urine sample, a chemically-adulterated urine sample, a lyophilized urine sample, a diluted natural urine sample, or a synthetic urine sample.
 12. A method of characterizing a urine sample from a subject, the method comprising: (a) providing a urine sample collected from a subject; (b) determining the absorbance at a first wavelength (A1) and the absorbance at a second wavelength (A2) of the urine sample, wherein the first wavelength is from about 230 nm to 250 nm, and the second wavelength is from about 260 nm to 340 nm; (c) applying an algorithm to the determined A1 and the determined A2 to generate a Urine Characterization Score (UCS), wherein the algorithm includes a ratio of the determined A1 to the determined A2; and (d) characterizing a urine sample as a natural urine sample, a chemically-adulterated urine sample, a lyophilized urine sample, a diluted natural urine sample, or a synthetic urine sample based on the UCS.
 13. The method of claim 12, wherein the UCS is generated using the Formula (XI): UCS=A1/A2.
 14. The method of claim 12, wherein the UCS is generated using the Formula (XII): UCS=10×log(A1/A2).
 15. The method of claim 12, wherein the first wavelength is 240 nm and the second wavelength is 280 nm.
 16. The method of claim 13, wherein step (d) comprises charactering the urine sample as a synthetic urine sample if UCS is greater than 0.8.
 17. The method of claim 14, wherein step (d) comprises charactering the urine sample as a synthetic urine sample if the UCS is less than 0.25.
 18. The method of claim 1, wherein the urine sample is characterized as a natural urine sample and the method further comprises: (e) performing an assay to determine the level of one or more drug metabolites in the urine sample.
 19. The method of claim 18, further comprising: (f) identifying a subject having an elevated level of one or more drug metabolites in the urine sample as compared to a reference level of the one or more drug metabolites, where the drug metabolites are metabolites of an illegal or controlled substance; and (g) admitting the subject into a drug dependency program, ceasing administration of the controlled substance to the subject, or reducing the dose and/or frequency of administration of the controlled substance to the subject.
 20. The method of claim 1, further comprising: (e) selecting a subject having a urine sample characterized in step (d) as a surfactant-adulterated urine sample, a lyophilized urine sample, a diluted natural urine sample, or a synthetic urine sample; and (f) obtaining an additional urine sample from the selected subject. 